Exome Sequencing in a Family with Luminal-Type Breast Cancer Underpinned by Variation in the Methylation Pathway.

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.

Genomic medicine and risk prediction across the disease spectrum.

Genomic medicine is based on the knowledge that virtually every medical condition, disease susceptibility or response to treatment is caused, regulated or influenced by genes. Genetic testing may therefore add value across the disease spectrum, ranging from single-gene disorders with a Mendelian inheritance pattern to complex multi-factorial diseases. The critical factors for genomic risk prediction are to determine: (1) where the genomic footprint of a particular susceptibility or dysfunction resides within this continuum, and (2) to what extent the genetic determinants are modified by environmental exposures. Regarding the small subset of highly penetrant monogenic disorders, a positive family history and early disease onset are mostly sufficient to determine the appropriateness of genetic testing in the index case and to inform pre-symptomatic diagnosis in at-risk family members. In more prevalent polygenic non-communicable diseases (NCDs), the use of appropriate eligibility criteria is required to ensure a balance between benefit and risk. An additional screening step may therefore be necessary to identify individuals most likely to benefit from genetic testing. This need provided the stimulus for the development of a pathology-supported genetic testing (PSGT) service as a new model for the translational implementation of genomic medicine in clinical practice. PSGT is linked to the establishment of a research database proven to be an invaluable resource for the validation of novel and previously described gene-disease associations replicated in the South African population for a broad range of NCDs associated with increased cardio-metabolic risk. The clinical importance of inquiry concerning family history in determining eligibility for personalized genotyping was supported beyond its current limited role in diagnosing or screening for monogenic subtypes of NCDs. With the recent introduction of advanced microarray-based breast cancer subtyping, genetic testing has extended beyond the genome of the host to also include tumor gene expression profiling for chemotherapy selection. The decreasing cost of next generation sequencing over recent years, together with improvement of both laboratory and computational protocols, enables the mapping of rare genetic disorders and discovery of shared genetic risk factors as novel therapeutic targets across diagnostic boundaries. This article reviews the challenges, successes, increasing inter-disciplinary integration and evolving strategies for extending PSGT towards exome and whole genome sequencing (WGS) within a dynamic framework. Specific points of overlap are highlighted between the application of PSGT and exome or WGS, as the next logical step in genetically uncharacterized patients for whom a particular disease pattern and/or therapeutic failure are not adequately accounted for during the PSGT pre-screen. Discrepancies between different next generation sequencing platforms and low concordance among variant-calling pipelines caution against offering exome or WGS as a stand-alone diagnostic approach. The public reference human genome sequence (hg19) contains minor alleles at more than 1 million loci and variant calling using an advanced major allele reference genome sequence is crucial to ensure data integrity. Understanding that genomic risk prediction is not deterministic but rather probabilistic provides the opportunity for disease prevention and targeted treatment in a way that is unique to each individual patient.

Understanding rural clinical learning spaces: Being and becoming a doctor.

CONTEXT: Calls for health professions education that can foster transformative educational experiences have been voiced. Studies suggest that extended clinical training at rural sites potentially provides transformative learning spaces. This article explores 'being and becoming' as a construct for understanding the student experience at a rural clinical school (RCS). METHODS: Sixty-two in-depth interviews were conducted over a three-year period with RCS students, graduates (as interns) and intern supervisors. Thematically analysed data were mapped according to the adapted Kirkpatrick model for appraising educational interventions. Drawing on realist perspectives, findings were further analysed to discern the mechanisms influencing the being and becoming of junior doctors. RESULTS: Responses provided evidence of changed attitudes and behaviour, and the adoption of professional practice that was seen to influence patient outcomes. Analysis highlighted sharing of values through role modelling, engagement with preceptors, being respected as part of a team, and being trusted to assume responsibility for a patient as key mechanisms. The outcome was confident, competent and caring interns. DISCUSSION: Rural clinical learning spaces influence the 'being and becoming' of a junior doctor. Understanding this process in the context of place (rural platform), participation (community of practice) and person can inform expanded agendas for students' clinical learning.

The impact of a faculty development programme for health professions educators in sub-Saharan Africa: an archival study.

BACKGROUND: In 2008 the sub-Saharan FAIMER Regional Institute launched a faculty development programme aimed at enhancing the academic and research capacity of health professions educators working in sub-Saharan Africa. This two-year programme, a combination of residential and distance learning activities, focuses on developing the leadership, project management and programme evaluation skills of participants as well as teaching the key principles of health professions education-curriculum design, teaching and learning and assessment. Participants also gain first-hand research experience by designing and conducting an education innovation project in their home institutions. This study was conducted to determine the perceptions of participants regarding the personal and professional impact of the SAFRI programme. METHODS: A retrospective document review, which included data about fellows who completed the programme between 2008 and 2011, was performed. Data included fellows' descriptions of their expectations, reflections on achievements and information shared on an online discussion forum. Data were analysed using Kirkpatrick's evaluation framework. RESULTS: Participants (n=61) came from 10 African countries and included a wide range of health professions educators. Five key themes about the impact of the SAFRI programme were identified: (1) belonging to a community of practice, (2) personal development, (3) professional development, (4) capacity development, and (5) tools/strategies for project management and/or advancement. CONCLUSION: The SAFRI programme has a positive developmental impact on both participants and their respective institutions.

Alienation and engagement in postgraduate training at a South African medical school.

BACKGROUND: The importance of contextual factors, such as the learning environment and sociocultural characteristics of the student, are becoming increasingly evident. Mann [2001. Alternative perspectives on the student experience: Alienation and engagement. Stud High Educ 26(1):7-19.] proposed that all learning experiences can be viewed as either alienating or engaging and Case expanded on this work. AIM: The purpose of this study was to explore perceptions of alienation or engagement as experienced by residents in anatomical pathology at one South African university. METHOD: A cross-sectional case study, with 16 semi-structured interviews was conducted. Residents were categorised as either alienated or engaged, based mainly on workplace experiences. RESULTS: Four relevant dimensions were identified; individual, home, workplace and institution. The personal attributes, strategies for coping and reasons for choosing pathology of alienated residents differed from those with engaged experiences. Poor socioeconomic background and schooling did not lead to predominantly alienating experiences, but this group still lacked some generic skills. In the workplace, two main factors resulting in alienated experiences were the interaction between residents and consultants and residents' comprehension of workplace-based learning. CONCLUSIONS: We present a simple model which may be used to identify factors that engage and alienate students in the learning experience in the workplace-based setting. Addressing these factors can contribute towards a more engaging experience for all residents.

Localisation of mycobacterial DNA and mRNA in human tuberculous granulomas.

In situ hybridisation was used to detect the presence of Mycobacterium tuberculosis in paraffin-embedded lung tissue of nine patients diagnosed with tuberculosis (TB). Mycobacterial DNA was found in all nine patients and in 175 out of 191 granulomas examined. A combination of in situ hybridisation and immunohistochemistry techniques demonstrated that mycobacterial DNA was associated with CD68-positive cells with the morphology of macrophages and giant cells. Mycobacterial DNA was also found within the necrotic regions of some granulomas. mRNA for the mycobacterial RNA polymerase beta subunit (rpoB) was detected by RNA: RNA in situ hybridisation. The rpoB mRNA was also localised to CD68-positive cells with the morphology of macrophages and to giant cells of certain necrotic granulomas. No rpoB mRNA was found in the necrotic regions of granulomas. Mycobacterial DNA was detected in 92% of patient granulomas of which 8% were positive for rpoB mRNA. The ability to identify mycobacterial RNA transcripts within human tuberculous granulomas affords us the opportunity to analyse the interplay between pathogen gene expression and the human immune response and should provide valuable insight into the mechanisms used by M. tuberculosis to persist within the human host.

Minimal supervision out-patient clinical teaching.

BACKGROUND: Minimal faculty member supervision of students refers to a method of instruction in which the patient-student encounter is not directly supervised by a faculty member, and presents a feasible solution in clinical teaching. It is unclear, however, how such practices are perceived by patients and how they affect student learning. CONTEXT: We aimed to assess patient and medical student perceptions of clinical teaching with minimal faculty member supervision. Questionnaires focusing on the perception of students' performance were administered to patients pre- and post-consultation. Students' self-perceptions on their performance were obtained using a questionnaire at the end of the consultation. INNOVATION: Before encounters with students, 22 per cent of the 95 patients were not sure if they would feel comfortable or trust the students; after the consultation, almost all felt comfortable (97%) and relied on the students (99%). The 81 students surveyed agreed that instruction with minimal faculty member supervision encouraged their participation and engagement (86%). They expressed interest in knowing patients' opinions about their performance (94%), and they felt comfortable about being assessed by the patients (86%). IMPLICATIONS: The minimal faculty member supervision model was well accepted by patients. Responses from the final-year students support the use of assessments that incorporate feedback from patients in their overall clinical evaluations.

Necrotizing enterocolitis in HIV-exposed and nonexposed infants: clinical presentation and histopathological features.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates and is associated with significant morbidity and mortality. An association between HIV-positive maternal status and increased risk of NEC in preterm infants has been described, and antiretroviral therapy has been proposed as an independent risk factor. Our aim was to compare the clinical presentation and histopathological features of necrotizing enterocolitis in HIV-exposed and unexposed infants. A retrospective study of archival material from the National Health Laboratory Services Histopathology Laboratory in Tygerberg Hospital/Stellenbosch University from 1992 to 2008 was conducted. All surgical specimens from infants who presented to pediatric surgery for a laparotomy and bowel resection for NEC and in whom the HIV status was known were included in the study. In the 37 cases that fulfilled these criteria, male gender was overrepresented in the study population (67%). Nonsteroidal anti-inflammatory drugs appeared to play a significant role in the development of surgical NEC in infants who were not exposed to HIV, but HIV-exposed infants had a significantly poorer survival rate. There was no significant difference in the histopathology between HIV-exposed and nonexposed infants, and Cytomegalovirus infection was not identified in any of the cases studied.

Microscopic colitis as a missed cause of chronic diarrhea.

AIM: To determine the prevalence of increased intraepithelial lymphocytes, using immunohistochemistry in patients with normal colonoscopy and near normal biopsy. METHODS: We retrospectively reviewed all non-malignant colon mucosal biopsies between 2005 and 2007, reported as normal, chronic inflammation or melanosis coli in patients who were undergoing routine colonoscopy. Immunohistochemistry using CD3 was performed on all mucosal biopsies and an intraepithelial lymphocyte count (IEL) was determined. Cases with an IEL count of ≥ 20 IELs per 100 surface epithelial cells were correlated with demographic, clinical and follow-up data. A further subgroup was evaluated for lymphocytic colitis. RESULTS: Twenty (8.3%) of 241 cases revealed an IEL count ≥ 20. Six (2.5%) patients were identified as having lymphocytic colitis (P < 0.001), of whom, five were missed on initial evaluation (P = 0.01). Four of these five patients were labeled with diarrhea-predominant irritable bowel syndrome (IBS). On follow-up, three of the remaining 20 cases were diagnosed with malignancy (renal cell carcinoma and myelodysplastic syndrome) and one had an unknown primary tumor with multiple liver metastases. Two cases of collagenous colitis with an IEL count < 10 were included in this study. Increased IELs were not confined to patients with diarrhea as a primary presenting symptom, but were also present in patients with abdominal pain (n = 7), constipation (n = 3) and loss of weight (n = 1). CONCLUSION: Immunohistochemistry using CD3 is of value in identifying and quantifying IELs for the presence of microscopic colitis in patients with diarrhea-predominant IBS.

Pleural tuberculosis in patients with early HIV infection is associated with increased TNF-alpha expression and necrosis in granulomas.

Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-gamma, TNF-alpha and IL-4 by in situ hybridisation. In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-alpha and necrosis. This study demonstrates immune dysregulation which may include TNF-alpha-mediated immunopathology at the site of disease in HIV infected pleural TB patients.

Associations between toll-like receptors and interleukin-4 in the lungs of patients with tuberculosis.

Toll-like receptors (TLRs) are implicated in the intracellular killing of Mycobacterium tuberculosis, and their expression is modulated by interleukin-4 (IL-4) in vitro. Our aim was to examine the expression of TLRs at the site of pathology in tuberculous lung granulomas and to explore the effect of the immune response on TLR expression. Immunohistochemistry was performed on lung granulomas from nine patients with tuberculosis undergoing lobectomy for haemoptysis. All nine patients expressed all of the TLRs studied (TLRs 1-5 and 9), whereas only five out of the nine patients had any granulomas positive for IL-4. Statistical analysis of TLR and cytokine staining patterns in 183 individual granulomas from the nine patients revealed significant associations between pairs of receptors and IL-4. A positive association between TLR2 and TLR4 (P < 0.0001) and a negative association between TLR2 and IL-4 (P < 0.0001) was observed. The associations between TLRs 1, 5, and 9 were significantly different in IL-4-negative compared with IL-4-positive patients. In conclusion, TLRs are expressed by various cell types in the human tuberculous lung, and their expression patterns are reflected by differences in the immune response.

Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas.

In order to examine the immune response at the site of pathology in tuberculosis, we analysed cytokines present in lung granulomas, their associations with each other and with caseous necrosis as well as the phenotype of the cellular infiltrate. Paraffin-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-gamma, TNF-alpha and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-alpha, IFN-gamma and IL-4 protein. Furthermore, CD68-positive myeloid cells expressed IL-12p40 mRNA, as expected, but a subset of CD3-positive lymphocytes also expressed this mRNA. These lymphocytes producing IL-12p40 also stained positive for CD8 but not CD4. A total of 141 granulomas were scored for the presence or absence of cytokine or necrosis and two major associations were identified. The first association was between IFN-gamma and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-gamma. The second association was between TNF-alpha and caseous necrosis, where all necrotic granulomas were TNF-alpha positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular infiltrate and cytokine expression is observed between adjacent granulomas in the same patient.

In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions.

We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host.