Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Can infant lung function predict respiratory morbidity during the first year of life in preterm infants?

Compared with term-born infants, preterm infants have increased respiratory morbidity in the first year of life. We investigated whether lung function tests performed near term predict subsequent respiratory morbidity during the first year of life and compared this to standard clinical parameters in preterms.The prospective birth cohort included randomly selected preterm infants with and without bronchopulmonary dysplasia. Lung function (tidal breathing and multiple-breath washout) was measured at 44 weeks post-menstrual age during natural sleep. We assessed respiratory morbidity (wheeze, hospitalisation, inhalation and home oxygen therapy) after 1 year using a standardised questionnaire. We first assessed the association between lung function and subsequent respiratory morbidity. Secondly, we compared the predictive power of standard clinical predictors with and without lung function data.In 166 preterm infants, tidal volume, time to peak tidal expiratory flow/expiratory time ratio and respiratory rate were significantly associated with subsequent wheeze. In comparison with standard clinical predictors, lung function did not improve the prediction of later respiratory morbidity in an individual child.Although associated with later wheeze, noninvasive infant lung function shows large physiological variability and does not add to clinically relevant risk prediction for subsequent respiratory morbidity in an individual preterm.

Prevalence of paunch calf syndrome carriers in Italian Romagnola cattle.

The term 'paunch calf syndrome' encompasses the multi-organic lethal developmental dysplasia reported in the Romagnola breed of cattle and is characterised by facial deformities, an enlarged and floating abdomen containing considerable abdominal effusion, and hepatic fibrosis. Paunch calf syndrome is caused by a missense mutation in the KDM2B gene (c.2503G>A) that is thought to lead to an amino acid exchange (p.D835N). In this study, the prevalence of carriers of the mutant KDM2B allele (and thus the frequency of the allele) was assessed in selected subpopulations of Romagnola cattle. The prevalence of carriers within top-ranked Romagnola sires over the years 2007-2012 was 29.3% (allele frequency 14.6%). In young bull calves, 30.9% were carriers with an allele frequency of 15.4%.

Head Reduction Osteotomy With Additional Containment Surgery Improves Sphericity and Containment and Reduces Pain in Legg-Calvé-Perthes Disease.

BACKGROUND Severe femoral head deformities in the frontal plane such as hips with Legg-Calvé-Perthes disease (LCPD) are not contained by the acetabulum and result in hinged abduction and impingement. These rare deformities cannot be addressed by resection, which would endanger head vascularity. Femoral head reduction osteotomy allows for reshaping of the femoral head with the goal of improving head sphericity, containment, and hip function. QUESTIONS/PURPOSES Among hips with severe asphericity of the femoral head, does femoral head reduction osteotomy result in (1) improved head sphericity and containment; (2) pain relief and improved hip function; and (3) subsequent reoperations or complications? METHODS Over a 10-year period, we performed femoral head reduction osteotomies in 11 patients (11 hips) with severe head asphericities resulting from LCPD (10 hips) or disturbance of epiphyseal perfusion after conservative treatment of developmental dysplasia (one hip). Five of 11 hips had concomitant acetabular containment surgery including two triple osteotomies, two periacetabular osteotomies (PAOs), and one Colonna procedure. Patients were reviewed at a mean of 5 years (range, 1-10 years), and none was lost to followup. Mean patient age at the time of head reduction osteotomy was 13 years (range, 7-23 years). We obtained the sphericity index (defined as the ratio of the minor to the major axis of the ellipse drawn to best fit the femoral head articular surface on conventional anteroposterior pelvic radiographs) to assess head sphericity. Containment was assessed evaluating the proportion of patients with an intact Shenton's line, the extrusion index, and the lateral center-edge (LCE) angle. Merle d'Aubigné-Postel score and range of motion (flexion, internal/external rotation in 90° of flexion) were assessed to measure pain and function. Complications and reoperations were identified by chart review. RESULTS At latest followup, femoral head sphericity (72%; range, 64%-81% preoperatively versus 85%; range, 73%-96% postoperatively; p = 0.004), extrusion index (47%; range, 25%-60% versus 20%; range, 3%-58%; p = 0.006), and LCE angle (1°; range, -10° to 16° versus 26°; range, 4°-40°; p = 0.0064) were improved compared with preoperatively. With the limited number of hips available, the proportion of an intact Shenton's line (64% versus 100%; p = 0.087) and the overall Merle d'Aubigné-Postel score (14.5; range, 12-16 versus 15.7; range, 12-18; p = 0.072) remained unchanged at latest followup. The Merle d'Aubigné-Postel pain subscore improved (3.5; range, 1-5 versus 5.0; range, 3-6; p = 0.026). Range of motion was not observed to have improved with the numbers available (p ranging from 0.513 to 0.778). In addition to hardware removal in two hips, subsequent surgery was performed in five of 11 hips to improve containment after a mean interval of 2.3 years (range, 0.2-7.5 years). Of those, two hips had triple osteotomy, one hip a combined triple and valgus intertrochanteric osteotomy, one hip an intertrochanteric varus osteotomy, and one hip a PAO with a separate valgus intertrochanteric osteotomy. No avascular necrosis of the femoral head occurred. CONCLUSIONS Femoral head reduction osteotomy can improve femoral head sphericity. Improved head containment in these hips with an often dysplastic acetabulum requires additional acetabular containment surgery, ideally performed concomitantly. This can result in reduced pain and avascular necrosis seems to be rare. With the number of patients available, function did not improve. Therefore, future studies should use more precise instruments to evaluate clinical outcome and include longer followup to confirm joint preservation. LEVEL OF EVIDENCE Level IV, therapeutic study.

Size and shape of the lunate surface in different types of pincer impingement: theoretical implications for surgical therapy.

OBJECTIVE Acetabular rim trimming is indicated in pincer hips with an oversized lunate surface but could result in a critically decreased size of the lunate surface in pincer hips with acetabular malorientation. There is a lack of detailed three-dimensional anatomy of lunate surface in pincer hips. Therefore, we questioned how does (1) size and (2) shape of the lunate surface differ among hips with different types of pincer impingement? METHOD We retrospectively compared size and shape of the lunate surface between acetabular retroversion (48 hips), deep acetabulum (34 hips), protrusio acetabuli (seven hips), normal acetabuli (30 hips), and hip dysplasia (45 hips). Using magnetic resonance imaging (MRI) arthrography with radial slices we measured size in percentage of the femoral head coverage and shape using the outer (inner) center-edge angles and width of lunate surface. RESULTS Hips with retroversion had a decreased size and deep hips had normal size of the lunate surface. Both had a normal shape of the outer acetabular rim. Protrusio hips had an increased size and a prominent outer acetabular rim. In all three types of pincer hips the acetabular fossa was increased. CONCLUSION Size and shape of the lunate surface differs substantially among different types of pincer impingement. In contrast to hips with protrusio acetabuli, retroverted and deep hips do not have an increased size of the lunate surface. Acetabular rim trimming in retroverted and deep hips should be performed with caution. Based on our results, acetabular reorientation would theoretically be the treatment of choice in retroverted hips.

Defined morphological criteria allow reliable diagnosis of colorectal serrated polyps and predict polyp genetics.

Criteria for the diagnosis of serrated colorectal lesions (hyperplastic polyp, sessile serrated adenoma without or with dysplasia--which we called mixed polyp--and traditional serrated adenoma) for which consensus has been reached should be validated for applicability in daily practice in terms of inter-observer reproducibility and their association with clinical features and (epi)genetic events. A study set was created from a consecutive series of colorectal polyps (n = 1,926) by selecting all sessile serrated adenomas, traditional serrated adenomas and mixed polyps. We added consecutive series of hyperplastic polyps, classical adenomas and normal mucosa samples for a total of 200 specimens. With this series, we conducted an inter-observer study, encompassing ten pathologists with gastrointestinal pathology experience from five European countries, in three rounds in which all cases were microscopically evaluated. An assessment of single morphological criteria was included, and these were correlated with clinical parameters and the mutation status of KRAS, BRAF and PIK3CA and the methylation status of MLH1. Gender, age and localisation were significantly associated with certain types of lesions. Kappa statistics revealed moderate to good inter-observer agreement for polyp classification (κ = 0.56 to 0.63), but for single criteria, this varied considerably (κ = 0.06 to 0.82). BRAF mutations were frequently found in hyperplastic polyps (86 %, 62/72) and sessile serrated adenomas (80 %, 41/51). KRAS mutations occurred more frequently in traditional serrated adenomas (78 %, 7/9) and less so in classical adenomas (20 %, 10/51). Single morphological criteria for sessile serrated adenomas showed significant correlation with BRAF mutation (all p ≤ 0.001), and those for classical adenomas or traditional serrated adenoma correlated significantly with KRAS mutation (all p < 0.001). Therefore, single well-defined morphological criteria are predictive for genetic alterations in colorectal polyps.

Surgical treatments for esophageal cancers.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the role of the nurse in preparation of esophageal resection (ER); the management of patients who develop high-grade dysplasia after having undergone Nissen fundoplication; the trajectory of care for the patient with esophageal cancer; the influence of the site of tumor in the choice of treatment; the best location for esophagogastrostomy; management of chylous leak after esophagectomy; the optimal approach to manage thoracic esophageal leak after esophagectomy; the choice for operational approach in surgery of cardioesophageal crossing; the advantages of robot esophagectomy; the place of open esophagectomy; the advantages of esophagectomy compared to definitive chemoradiotherapy; the pathologist report in the resected specimen; the best way to manage patients with unsuspected positive microscopic margin after ER; enhanced recovery after surgery for ER: expedited care protocols; and long-term quality of life in patients following esophagectomy.

Multi-colour FISH in oesophageal adenocarcinoma-predictors of prognosis independent of stage and grade.

BACKGROUND Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples. METHODS To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data. RESULTS The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months. CONCLUSIONS Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.

Implications of Imaging Criteria for the Management and Treatment of Intraductal Papillary Mucinous Neoplasms - Benign versus Malignant Findings

OBJECTIVES Evaluation of computed tomography (CT) and magnetic resonance imaging (MRI) for differentiation of pancreatic intraductal papillary mucinous neoplasm (IPMN) subtypes based on objective imaging criteria. METHODS Fifty-eight patients with 60 histologically confirmed IPMNs were included in this retrospective study. Eighty-three imaging studies (CT,n = 42; MRI,n = 41) were analysed by three independent blinded observers (O1-O3), using established imaging criteria to assess likelihood of malignancy (-5, very likely benign; 5, very likely malignant) and histological subtype (i.e., low-grade (LGD), moderate-grade (MGD), high-grade dysplasia (HGD), early invasive carcinoma (IPMC), solid carcinoma (CA) arising from IPMN). RESULTS Forty-one benign (LGD IPMN,n = 20; MGD IPMN,n = 21) and 19 malignant (HGD IPMN,n = 3; IPMC,n = 6; solid CA,n = 10) IPMNs located in the main duct (n = 6), branch duct (n = 37), or both (n = 17) were evaluated. Overall accuracy of differentiation between benign and malignant IPMNs was 86/92 % (CT/MRI). Exclusion of overtly malignant cases (solid CA) resulted in overall accuracy of 83/90 % (CT/MRI). The presence of mural nodules and ductal lesion size ≥30 mm were significant indicators of malignancy (p = 0.02 and p < 0.001, respectively). CONCLUSIONS Invasive IPMN can be identified with high confidence and sensitivity using CT and MRI. The diagnostic problem that remains is the accurate radiological differentiation of premalignant and non-invasive subtypes. KEY POINTS • CT and MRI can differentiate benign from malignant forms of IPMN. • Identifying (pre)malignant histological IPMN subtypes by CT and MRI is difficult. • Overall, diagnostic performance with MRI was slightly (not significantly) superior to CT.

Preliminary Study on Finite Element Simulation for Optimizing Acetabulum Reorientation after Periacetabular Osteotomy

Periacetabular osteotomy (PAO) is an effective approach for surgical treatment of hip dysplasia. The aim of PAO is to increase acetabular coverage of the femoral head and to reduce contact pressures by reorienting the acetabulum fragment after PAO. The success of PAO significantly depends on the surgeon’s experience. Previously, we have developed a computer-assisted planning and navigation system for PAO, which allows for not only quantifying the 3D hip morphology for a computer-assisted diagnosis of hip dysplasia but also a virtual PAO surgical planning and simulation. In this paper, based on this previously developed PAO planning and navigation system, we developed a 3D finite element (FE) model to investigate the optimal acetabulum reorientation after PAO. Our experimental results showed that an optimal position of the acetabulum can be achieved that maximizes contact area and at the same time minimizes peak contact pressure in pelvic and femoral cartilages. In conclusion, our computer-assisted planning and navigation system with FE modeling can be a promising tool to determine the optimal PAO planning strategy.

What Are the Radiographic Reference Values for Acetabular Under- and Overcoverage?

Background Both acetabular undercoverage (hip dysplasia) and overcoverage (pincer-type femoroacetabular impingement) can result in hip osteoarthritis. In contrast to undercoverage, there is a lack of information on radiographic reference values for excessive acetabular coverage. Questions/purposes (1) How do common radiographic hip parameters differ in hips with a deficient or an excessive acetabulum in relation to a control group; and (2) what are the reference values determined from these data for acetabular under- and overcoverage? Methods We retrospectively compared 11 radiographic parameters describing the radiographic acetabular anatomy among hip dysplasia (26 hips undergoing periacetabular osteotomy), control hips (21 hips, requiring no rim trimming during surgical hip dislocation), hips with overcoverage (14 hips, requiring rim trimming during surgical hip dislocation), and hips with severe overcoverage (25 hips, defined as having acetabular protrusio). The hips were selected from a patient cohort of a total of 593 hips. Radiographic parameters were assessed with computerized methods on anteroposterior pelvic radiographs and corrected for neutral pelvic orientation with the help of a true lateral radiograph. Results All parameters except the crossover sign differed among the four study groups. From dysplasia through control and overcoverage, the lateral center-edge angle, acetabular arc, and anteroposterior/craniocaudal coverage increased. In contrast, the medial center-edge angle, extrusion/acetabular index, Sharp angle, and prevalence of the posterior wall sign decreased. The following reference values were found: lateral center-edge angle 23° to 33°, medial center-edge angle 35° to 44°, acetabular arc 61° to 65°, extrusion index 17% to 27%, acetabular index 3° to 13°, Sharp angle 38° to 42°, negative crossover sign, positive posterior wall sign, anterior femoral head coverage 15% to 26%, posterior femoral head coverage 36% to 47%, and craniocaudal coverage 70% to 83%. Conclusions These acetabular reference values define excessive and deficient coverage. They may be used for radiographic evaluation of symptomatic hips, may offer possible predictors for surgical outcomes, and serve to guide clinical decision-making.

A mutation in hairless dogs implicates FOXI3 in ectodermal development

Mexican and Peruvian hairless dogs and Chinese crested dogs are characterized by missing hair and teeth, a phenotype termed canine ectodermal dysplasia (CED). CED is inherited as a monogenic autosomal semidominant trait. With genomewide association analysis we mapped the CED mutation to a 102-kilo-base pair interval on chromosome 17. The associated interval contains a previously uncharacterized member of the forkhead box transcription factor family (FOXI3), which is specifically expressed in developing hair and teeth. Mutation analysis revealed a frameshift mutation within the FOXI3 coding sequence in hairless dogs. Thus, we have identified FOXI3 as a regulator of ectodermal development.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

BACKGROUND Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Ectodysplasin-1 deficiency in a German Holstein bull associated with loss of respiratory mucous glands and chronic rhinotracheitis

A 2-year-old German Holstein bull was identified as a carrier of a mutation within the X-chromosomal ED1 gene, which encodes a TNF-related signalling molecule mainly involved in ectodermal development. The clinicopathological appearance was associated with hypotrichosis, hypodontia, and a reduced number of eccrine glands, in addition to chronic rhinotracheitis and partial squamous metaplasia. Furthermore, for the first time in an ED1-deficient animal, a complete lack of respiratory mucous glands was observed. This suggests that the ED1 gene plays a role in the development of mucous glands, the absence of which resembles a feature of X-linked anhidrotic ectodermal dysplasia (ED1) in human patients.

An Insight Into Cervical Cancer Screening and Treatment Capacity in Sub Saharan Africa.

OBJECTIVE Approximately 85% of cervical cancer cases and deaths occur in resource-constrained countries where best practices for prevention, particularly for women with HIV infection, still need to be developed. The aim of this study was to assess cervical cancer prevention capacity in select HIV clinics located in resource-constrained countries. MATERIALS AND METHODS A cross-sectional survey of sub-Saharan African sites of 4 National Institutes of Health-funded HIV/AIDS networks was conducted. Sites were surveyed on the availability of cervical cancer screening and treatment among women with HIV infection and without HIV infection. Descriptive statistics and χ or Fisher exact test were used as appropriate. RESULTS Fifty-one (65%) of 78 sites responded. Access to cervical cancer screening was reported by 49 sites (96%). Of these sites, 39 (80%) performed screening on-site. Central African sites were less likely to have screening on-site (p = .02) versus other areas. Visual inspection with acetic acid and Pap testing were the most commonly available on-site screening methods at 31 (79%) and 26 (67%) sites, respectively. High-risk HPV testing was available at 29% of sites with visual inspection with acetic acid and 50% of sites with Pap testing. Cryotherapy and radical hysterectomy were the most commonly available on-site treatment methods for premalignant and malignant lesions at 29 (74%) and 18 (46%) sites, respectively. CONCLUSIONS Despite limited resources, most sites surveyed had the capacity to perform cervical cancer screening and treatment. The existing infrastructure of HIV clinical and research sites may provide the ideal framework for scale-up of cervical cancer prevention in resource-constrained countries with a high burden of cervical dysplasia.