Brenda L. Gallie

Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression.

We have analyzed RNA from retinoblastoma patients and unaffected carriers with various RB1 gene mutations to determine the patterns of missplicing and associations with phenotypic expression. Most sequence alterations in or in the neighborhood of conserved splice signals that we tested resulted in simple exon skipping (15 mutations) or intron inclusion (new acceptor AG‐sites, four mutations) as expected. Two mutations resulted in skipping of a neighboring exon (exon 11), a complex pattern indicating competition for correct lariat formation. We observed no activation of a cryptic splice site but found that a recurrent missense mutation in exon 7 creates a new splice site (two families). RT‐PCR analysis enabled us to confirm the presence and to characterize the transcriptional consequences of gross insertions and deletions in the RB1 gene in six patients, including two patients with mutational mosaicism. We also used RT‐PCR analysis to search for unknown mutations in 15 patients and identified three oncogenic point mutations deep in introns. Two of these mutations are recurrent thus indicating that, despite the vast extent of the introns of the RB1 gene, few bases are effective targets for oncogenic mutations. When analyzing associations between phenotypic expression (16 families) and mutational consequences we observed no link to the presence or absence of a premature termination codon in the mutant transcript. However, the location of a mutation relative to the splice sequence has a strong and consistent influence on phenotypic expression. Hum Mutat 29(4), 475–484, 2008.

Nuclear E-cadherin and VHL immunoreactivity are prognostic indicators of clear-cell renal cell carcinoma

The loss of functional von Hippel–Lindau (VHL) tumor suppressor gene is associated with the development of clear-cell renal cell carcinoma (CC-RCC). Recently, VHL was shown to promote the transcription of E-cadherin, an adhesion molecule whose expression is inversely correlated with the aggressive phenotype of numerous epithelial cancers. Here, we performed immunohistochemistry on CC-RCC tissue microarrays to determine the prognostic value of E-cadherin and VHL with respect to Fuhrman grade and clinical prognosis. Low Fuhrman grade and good prognosis associated with positive VHL and E-cadherin immunoreactivity, whereas poor prognosis and high-grade tumors associated with a lack of E-cadherin and lower frequency of VHL staining. A significant portion of CC-RCC with positive VHL immunostaining correlated with nuclear localization of C-terminally cleaved E-cadherin. DNA sequencing revealed in a majority of nuclear E-cadherin-positive CC-RCC, subtle point mutations, deletions and insertions in VHL. Furthermore, nuclear E-cadherin was not observed in chromophobe or papillary RCC, as well as matched normal kidney tissue. In addition, nuclear E-cadherin localization was recapitulated in CC-RCC xenografts devoid of functional VHL or reconstituted with synthetic mutant VHL grown in SCID mice. These findings provide the first evidence of aberrant nuclear localization of E-cadherin in CC-RCC harboring VHL mutations, and suggest potential prognostic value of VHL and E-cadherin in CC-RCC.

KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

The kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules.

Global issues and opportunities for optimized retinoblastoma care.

The RB1 gene is important in all human cancers. Studies of human retinoblastoma point to a rare retinal cell with extreme dependency on RB1 for initiation but not progression to full malignancy. In developed countries, genetic testing within affected families can predict children at high risk of retinoblastoma before birth; chemotherapy with local therapy often saves eyes and vision; and mortality is 4%. In less developed countries where 92% of children with retinoblastoma are born, mortality reaches 90%. Global collaboration is building for the dramatic change in mortality that awareness, simple expertise and therapies could achieve in less developed countries. Pediatr Blood Cancer 2007;49:1083–1090.

Structured electronic operative reporting: Comparison with dictation in kidney cancer surgery

PURPOSE The purpose of this study was to evaluate the functionality of eKidney as a structured reporting tool in operative note generation. To do this, we compared completeness and timeliness of eKidney template-generated nephrectomy OR notes with standard narrative dictation. METHODS A group of academic uro-oncologists and medical informaticians at the University Health Network designed and adopted an electronic online, point-of-care clinical documentation tool, eCancerCare(Kidney) (eKidney) for kidney cancer patient care. The optimal components of clinic and operative note templates, including those for nephrectomy, were agreed upon by expert consensus of the uro-oncologists. Clinician nephrectomy OR reports were analyzed for completeness, comparing those generated in eKidney with conventionally dictated notes. Patterns of missing information from both dictated and eKidney-generated reports were analyzed. The procedure, note completion and transcription dates were recorded which generated time intervals between these events. The records of 189 procedures were included in the analysis. RESULTS Comparison of clinicians who used both note generation modalities, revealed a mean completion rate of 92% for eKidney/structured notes and 68% for dictated notes (p<0.0001). There was no significant difference in completion rates between attending staff and trainees (residents and fellows) (p=0.131). Most notes were dictated/entered on the day of surgery. Dictated notes were transcribed to EPR a median of 2 days after dictation, however roughly 30% of dictated notes took 5 days or more to get transcribed. All notes generated using eKidney were uploaded to the EPR immediately. LIMITATIONS Our study has three significant limitations. Firstly, our study was not randomized: physicians could elect to dictate or use eKidney. Secondly, we did not identify data from dictated notes that were not captured by eKidney. Third, we did not compare the time it took physicians to complete the fields in eKidney with the time it takes to dictate a note. CONCLUSIONS We have demonstrated that the use of structured reporting improves the completeness and timeliness of documentation in kidney cancer surgery. eKidney is an example of the power of templates in ensuring that important details of a procedure are recorded. Future studies looking at user satisfaction, and research and educational potential of eKidney would be valuable.

Identification of clinically relevant mosaicism in type I hereditary haemorrhagic telangiectasia

Background Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder affecting the vascular system, characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. Mutations in two genes, ENG and ACVRL1, account for the majority of cases. Almost all cases of HHT show a family history of HHT-associated symptoms; few cases are de novo. Mutational mosaicism is the presence of two populations of cells, with both mutant and normal genotypes in one individual and generally occurs through de novo mutation events in embryogenesis. Some isolated cases of HHT with no detectable ENG or ACVRL1 mutation may be caused by a mosaic ENG or ACVRL1 mutation that is present at levels below the limit of detection of current molecular screening methods. Objective To identify clinically relevant mosaicism in type I HHT. Methods Sequencing, quantitative multiplex-PCR and marker analysis were used to identify three HHT families with founders who showed mosaicism for endoglin mutations. Where available, mosaicism was verified by testing different sampling sites, including blood, hair and buccal swabs. Results All three mosaic samples exhibited the mutation in an estimated ≤25% of the DNA. Two of the mosaic patients had clinically confirmed HHT by the Curaçao criteria and the other showed symptoms of HHT. In each case the heterozygous mutation had already been identified in another family member before detection in the mosaic founder. Conclusions The results show the importance of investigating patients without prior family history for the presence of mutational mosaicism, as detecting this would enable appropriate genetic screening and targeted medical care for at-risk children of mosaic patients.