A novel nicastrin mutation in a three-generation Dutch family with hidradenitis suppurativa: a search for functional significance.

BACKGROUND: Mutations in the γ-secretase enzyme subunits have been described in multiple kindreds with familial hidradenitis suppurativa (HS). OBJECTIVE: In this study, we report a novel nicastrin (NCSTN) mutation causing HS in a Dutch family. We sought to explore the immunobiological function of NCSTN mutations using data of the Immunological Genome Project. METHODS: Blood samples of three affected and two unaffected family members were collected. Whole-genome sequencing was performed using genomic DNA isolated from peripheral blood leucocytes. Sanger sequencing was done to confirm the causative NCSTN variant and the familial segregation. The microarray data set of the Immunological Genome Project was used for thorough dissection of the expression and function of wildtype NCSTN in the immune system. RESULTS: In a family consisting of 23 members, we found an autosomal dominant inheritance pattern of HS and detected a novel splice site mutation (c.1912_1915delCAGT) in the NCSTN gene resulting in a frameshift and subsequent premature stop. All affected individuals had HS lesions on non-flexural and atypical locations. Wildtype NCSTN appears to be upregulated in myeloid cells like monocytes and macrophages, and in mesenchymal cells such as fibroblastic reticular cells and fibroblasts. In addition, within the 25 highest co-expressed genes with NCSTN we identified CAPNS1, ARNT and PPARD. CONCLUSION: This study reports the identification a novel NCSTN gene splice site mutation which causes familial HS. The associated immunobiological functions of NCSTN and its co-expressed genes ARNT and PPARD link genetics to the most common environmental and metabolic HS risk factors which are smoking and obesity.

MALDI-TOF MS meets WGS in a VRE outbreak investigation.

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.

The role of defensive ecological interactions in the evolution of conotoxins.

Venoms comprise of complex mixtures of peptides evolved for predation and defensive purposes. Remarkably, some carnivorous cone snails can inject two distinct venoms in response to predatory or defensive stimuli, providing a unique opportunity to study separately how different ecological pressures contribute to toxin diversification. Here, we report the extraordinary defensive strategy of the Rhizoconus subgenus of cone snails. The defensive venom from this worm-hunting subgenus is unusually simple, almost exclusively composed of αD-conotoxins instead of the ubiquitous αA-conotoxins found in the more complex defensive venom of mollusc- and fish-hunting cone snails. A similarly compartmentalized venom gland as those observed in the other dietary groups facilitates the deployment of this defensive venom. Transcriptomic analysis of a Conus vexillum venom gland revealed the αD-conotoxins as the major transcripts, with lower amounts of 15 known and four new conotoxin superfamilies also detected with likely roles in prey capture. Our phylogenetic and molecular evolution analysis of the αD-conotoxins from five subgenera of cone snails suggests they evolved episodically as part of a defensive strategy in the Rhizoconus subgenus. Thus, our results demonstrate an important role for defence in the evolution of conotoxins.

The Physical Activity and Health Status of Two Generations of Black South African Professional Women

Increased health risks associated with physical inactivity in the Black population have been reported in recent years. Black women; suffering the highest levels of inactivity; overweight and obesity; are at greatest risk of developing chronic diseases of lifestyle. This explorative-descriptive study investigated the physical activity patterns and health status of two generations of Black professional women; reflecting pre-democracy and post-democracy age groups. Quantitative measures were used; including the ActiGraph GT1M accelerometer; the Global Physical Activity Questionnaire and the Health-Promoting Lifestyle Profile. Sample groups comprised teachers; nurses; social workers and public sector managers. Participants aged between 35 and 45 years were allocated to the older generation group (n = 111); whilst those aged between 18 and 21 years (students in the mentioned professional fields) were allocated to the younger generation group (n = 69). The results indicated that these women displayed lower levels of health-promoting behavioural practices than expected; significantly lower levels of physical activity and significantly higher levels of overweight and obesity than the South African norms. The observation that the younger group appeared to be replicating the patterns of the older women is a cause of concern. Greater compliance to health-promoting behaviours was expected in this group owing to participants' professional involvement in health; education and social development fields. Wide-ranging initiatives are necessary to promote physical activity and health amongst the Black female population in South Africa

Public Perception of Community Pharmacists in South Africa : Primary Study

This survey forms part of a study undertaken to identify and quantify the community pharmacist's involvement in the use of non-prescription/over-the-counter (OTC) medicine /self-medication in community pharmacies throughout South Africa. The objectives of this study were to establish the public's perception of the role of and the value added by community pharmacists in the treatment of minor ailments. A survey was conducted amongst 300 consumers prior to the recent changes and introduction of new legislation affecting pharmaceutical care and the dispensing of medicine. The major finding of this study was that the majority of consumers who consulted community pharmacists were satisfied with the comprehensiveness of the counselling and the effectiveness of the treatment provided. A large majority of the participants agreed that community pharmacists play a positive role and add value. It was found that the main reason for visiting a specific pharmacy was the proximity of that pharmacy

Histology of the fascial-periosteal interface in lower limb chronic deep posterior compartment syndrome.

OBJECTIVE: To describe the histological features of the fascial-periosteal interface at the medial tibial border of patients surgically treated for chronic deep posterior compartment syndrome and to make statistical comparisons with control tissue. METHODS: Nineteen subjects and 11 controls were recruited. Subject tissue was obtained at operation, and control tissue from autopsy cases. Tissue samples underwent histological preparation and then examination by an independent pathologist. Samples were analysed with regard to six histological variables: fibroblastic activity, chronic inflammatory cells, vascularity, collagen regularity, mononuclear cells, and ground substance. Collagen regularity was measured with respect to collagen density, fibre arrangement, orientation, and spacing. The observed changes were graded from 1 to 4 in terms of abnormality. Mann-Whitney U test, Spearman correlation coefficients, and intraobserver reliability scores were used. RESULTS: With regard to collagen arrangement, control tissue showed greater degrees of irregularity than subject tissue (p = 0.01). Subjects with a symptom duration of greater than 12 months (as opposed to less than 12 months) showed greater degrees of collagen irregularity (p = 0.043). Vascular changes approached significance (p = 0.077). With regard to the amount of fibrocyte activity, chronic inflammatory cell activity, mononuclear cells, or ground substance, there were no significant differences between controls and subjects. Good correlation was seen in scores measuring chronic inflammatory cell activity and mononuclear cells (r = 0.649), and moderate correlation was seen between fibrocyte activity and vascular changes (r = 0.574). Intraobserver reliability scores were good for chronic inflammatory cell activity and moderate for vascular changes, but were poor for collagen and fibrocyte variables. Individual cases showed varying degrees of fibrocyte activity, chronic inflammatory cellular infiltration, vascular abnormalities, and collagen fibre disruption. CONCLUSIONS: Statistical analysis showed no histological differences at the fascial-periosteal interface in cases of chronic deep posterior compartment syndrome, except for collagen, which showed less irregularity in subject samples. The latter may indicate a remodelling process, and this is supported by greater collagen irregularity in subjects with longer duration of symptoms.

Risk factors for breast cancer in young women by oestrogen receptor and progesterone receptor status.

We used data from 765 cases and 564 controls in the population-based Australian Breast Cancer Family Study to investigate whether, in women under the age of 40, the profile of risk factors differed between breast cancer subtypes defined by joint oestrogen and progesterone receptor status. As hypothesised, no significant differences were found.

Searching for candidate genes for male infertility.

AIM: We describe an approach to search for candidate genes for male infertility using the two human genome databases: the public University of California at Santa Cruz (UCSC) and private Celera databases which list known and predicted gene sequences and provide related information such as gene function, tissue expression, known mutations and single nucleotide polymorphisms (SNPs). METHODS AND RESULTS: To demonstrate this in silico research, the following male infertility candidate genes were selected: (1) human BOULE, mutations of which may lead to germ cell arrest at the primary spermatocyte stage, (2) mutations of casein kinase 2 alpha genes which may cause globozoospermia, (3) DMR-N9 which is possibly involved in the spermatogenic defect of myotonic dystrophy and (4) several testes expressed genes at or near the breakpoints of a balanced translocation associated with hypospermatogenesis. We indicate how information derived from the human genome databases can be used to confirm these candidate genes may be pathogenic by studying RNA expression in tissue arrays using in situ hybridization and gene sequencing. CONCLUSION: The paper explains the new approach to discovering genetic causes of male infertility using information about the human genome.

A specific GFP expression assay, penetrance estimate, and histological assessment for a putative splice site mutation in BRCA1.

Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer.

Biological markers that predict clinical recurrence in ductal carcinoma in situ of the breast.

The optimal management of ductal carcinoma in situ (DCIS) is controversial, due in part to our poor understanding of its natural history. We undertook to identify subgroups of DCIS based on the expression of biomarkers, which were related to the likelihood of clinical recurrence. Biomarker expression of a total of 95 DCIS lesions in a nested case-control study within a population-based cohort with up to 135 months follow-up data (median 101 months) was analysed using immunohistochemistry. ERBB2-positivity and bcl-2-, oestrogen receptor (ER)- and progesterone receptor (PR)-negativity were individually associated with the risk of clinical recurrence. The predictive value of these biomarkers was independent of cytonuclear grade. ERBB2, bcl-2, ER and PR expression were conserved in the recurrent lesions, including subsequent invasive cancers. p21-positive DCIS was also associated with clinical recurrence, independently of the associations with ERBB2/bcl-2/ER/PR expression. These data identify clinically and biologically relevant subcategories of DCIS lesions, an essential basis for improving management.

The steroid 5alpha-reductase type II TA repeat polymorphism is not associated with risk of breast or ovarian cancer in Australian women.

The enzyme 5alpha-reductase type II (SRD5A2) converts testosterone to its more active form 5alpha-dihydroxytestosterone. The 3' untranslated region of the gene contains a (TA)(n) length polymorphism. The (TA)(9) allele has been reported to be associated with higher serum prostate-specific antigen levels in breast tumors and lower risk of relapse in breast cancer patients and more recently has also been reported to be linked to the codon 89 valine variant, which is itself associated with higher serum prostate-specific antigen levels in breast tumors and a more favorable breast cancer prognosis. We investigated whether the SRD5A2 (TA)(n) polymorphism was associated with risk of breast or ovarian cancer in Australian women by studying 946 breast cancer cases and 509 age-matched controls, and 544 ovarian cancer cases and 298 controls of similar age distribution. The (TA)(9) allele frequency was similar in breast cancer cases (0.110), breast cancer controls (0.125), ovarian cancer cases (0.106), and ovarian cancer controls (0.117). There was no difference in genotype distribution between breast cancer cases and controls (P = 0.5), ovarian cancer cases and controls (P = 0.7), or between the two control groups (P = 0.9). Genotypes containing at least one (TA)(9) allele were not significantly associated with risk of breast cancer overall (odds ratio, 0.86; 95% confidence interval, 0.67-1.12; P = 0.3) or in women stratified by age, menopausal status, or family history. Similarly, the (TA)(9) allele was not associated with risk of ovarian cancer (odds ratio, 0.87; 95% confidence interval, 0.61-1.23; P = 0.4) or with ovarian tumor behavior (invasive or low malignant potential), histology, stage, or grade. Given that this study had sufficient power to detect altered risks in the order of 1.4- to 1.7-fold, our results suggest that the SRD5A2 (TA)(9) allele is unlikely to be associated with moderate alterations in breast or ovarian cancer risk.

Gene targeting of Desrt, a novel ARID class DNA-binding protein, causes growth retardation and abnormal development of reproductive organs.

We have cloned and characterized a novel murine DNA-binding protein Desrt, with a motif characteristic of the ARID (A-T rich interaction domain) family of transcription factors. The Desrt gene encodes an 83-kD protein that is shown to bind DNA and is widely expressed in adult tissues. To examine the in vivo function of Desrt, we have generated mice with a targeted mutation in the ARID domain of Desrt. Homozygous mutants have reduced viability, pronounced growth retardation, and a high incidence of abnormalities of the female and male reproductive organs including cryptorchidism. This may thus serve as a model to dissect the mechanisms involved in the development of the reproductive tract including testicular descent. Gene-targeted mice also display a reduction in the thickness of the zona reticularis of the adrenal gland and transient aberrations of the T and B cell compartments of primary lymphoid organs. These data show that this novel DNA-binding protein, Desrt, has a nonredundant function during growth and in the development of the reproductive system.

Molecular pathologic analysis enhances the diagnosis and management of Muir-Torre syndrome and gives insight into its underlying molecular pathogenesis.

The Muir-Torre syndrome (MTS) is an autosomal dominantly inherited disorder, characterized by visceral malignancies and sebaceous skin lesions. In a subset of MTS families the disease is due to an underlying DNA mismatch-repair defect. We have identified a MTS family whose spectrum of reported neoplasia included adenocarcinomas of numerous gastrointestinal sites, carcinomas of the endometrium, ovary and breast, papillary transitional cell carcinoma of the ureter, a range of cutaneous tumors, as well as keratoacanthomas. All tumors were tested for microsatellite instability and immunohistochemically stained for expression of MLH1 and MSH2 proteins. All tumors were found to be microsatellite unstable and lacking in MSH2 protein expression. The subsequent mutation detection focused on hMSH2, and a germline mutation was identified (CAA-->TAA, Gln-->STOP, codon 337). This mutation was subsequently found in a family member with a single skin lesion only. We propose that the combination of immunohistologic and microsatellite instability analysis can be exploited to screen individuals with characteristic skin lesions even before development of visceral tumors and to direct the subsequent germline mutation search. The profile of microsatellite instability and the genes rendered dysfunctional differed between tumor samples, suggesting that the molecular pathogenesis varied between lesions, despite a common germline mutation.

Overexpression of the steroid receptor coactivator AIB1 in breast cancer correlates with the absence of estrogen and progesterone receptors and positivity for p53 and HER2/neu.

The gene for the steroid receptor coactivator amplified in breast cancer 1 (AIBI), located on chromosome 20q12, is overexpressed at the mRNA level in up to 60% of primary breast carcinomas; however, only 5% of these tumors show DNA amplification. The transcription factors and signaling pathways relevant to breast cancer, which in the absence of DNA amplification are responsible for and targeted by elevated levels of AIBI mRNA, are unknown. In the present study, in situ hybridization was used to examine AIB1 mRNA expression in 93 breast carcinomas of varying histological grade and immunohistochemical profile. AIB1 mRNA was overexpressed relative to normal breast tissue in 26 of 83 (31%) invasive tumors. This was found to associate with high tumor grade (P = 0.0006), lack of immunohistochemical staining for the steroid receptors estrogen receptor (P = 0.002) and progesterone receptor (P = 0.002), and strong protein staining for p53 (P = 0.01) and HER2/neu (P = 0.002). These findings suggest that AIB1 overexpression may impact on breast cancer by a mechanism not wholly dependent on steroid receptor coexpression and which may involve other oncogenic events, such as p53 protein stabilization and HER2/neu overexpression.

After BRCA1 and BRCA2-what next? Multifactorial segregation analyses of three-generation, population-based Australian families affected by female breast cancer.

Mutations in BRCA1 and BRCA2 that cause a dominantly inherited high risk of female breast cancer seem to explain only a small proportion of the aggregation of the disease. To study the possible additional genetic components, we conducted single-locus and two-locus segregation analyses, with and without a polygenic background, using three-generation families ascertained through 858 women with breast cancer diagnosed at age <40 years, ascertained through population cancer registries in Melbourne and Sydney, Australia. Extensive testing for deleterious mutations in BRCA1 and BRCA2, to date, has identified 34 carriers. Our analysis suggested that, after other possible unmeasured familial factors are adjusted for and the known BRCA1 and BRCA2 mutation carriers are excluded, there appears to be a residual dominantly inherited risk of female breast cancer in addition to that derived from mutations in BRCA1 and BRCA2. This study also suggests that there is a substantial recessively inherited risk of early-onset breast cancer. According to the best-fitting model, after excluding known carriers of mutations in BRCA1 and BRCA2, about 1/250 (95% confidence interval [CI] 1/500 to 1/125) women have a recessive risk of 86% (95% CI 69%-100%) by age 50 years and of almost 100% by age 60 years. Possible reasons that our study has implicated a novel strong recessive effect include our inclusion of data on lineal aunts and grandmothers, study of families ascertained through women with early-onset breast cancer, allowance for multiple familial factors in the analysis, and removal of families for whom the cause (i.e., BRCA1 or BRCA2) is known. Our findings may have implications for attempts to identify new breast cancer-susceptibility genes.

CYP17 promoter polymorphism and breast cancer in Australian women under age forty years.

BACKGROUND: The cytochrome P450c17alpha enzyme functions in the steroid biosynthesis pathway, and altered endogenous steroid hormone levels have been reported to be associated with a T to C polymorphism in the 5' promoter region of the CYP17 gene. Because steroid hormone exposure is known to influence breast cancer risk, we conducted a population-based, case-control-family study to assess the relationship between the CYP17 promoter polymorphism and early-onset breast cancer. METHODS: Case subjects under 40 years of age at diagnosis of a first primary breast cancer, population-sampled control subjects, and the relatives of both case and control subjects were interviewed to record family history of breast cancer and other risk factors. CYP17 genotype was determined in 369 case subjects, 284 control subjects, and 91 relatives of case subjects. Genotype distributions were compared by logistic regression, and cumulative risk was estimated by a modified segregation analysis. All statistical tests were two-tailed. RESULTS: Compared with the TT genotype (i.e., individuals homozygous for the T allele), the TC genotype was not associated with increased breast cancer risk (P: =.7). Compared with the TT and TC genotypes combined, the CC genotype was associated with a relative risk of 1. 81 (95% confidence interval [CI] = 1.15-2.86; P: =.01) before adjustment for measured risk factors and 1.63 (95% CI = 1.00-2.64; P: =.05) after adjustment. There was an excess of CC genotypes in case subjects who had at least one affected first- or second-degree relative, compared with control subjects unstratified by family history of breast cancer (23% versus 11%; P: =.006), and these case subjects had a threefold to fourfold higher risk than women of other groups defined by genotype and family history of breast cancer. Analysis of breast cancer in first- and second-degree relatives of case subjects with the CC genotype, excluding two known carriers of a deleterious mutation in BRCA1 or BRCA2, gave a relative hazard in women with the CC genotype of 3.48 (95% CI = 1.13-10.74; P: =.04), which is equivalent to a cumulative risk of 16% to age 70 years. CONCLUSIONS: The CC genotype may modify the effect of other familial risk factors for early-onset breast cancer.

Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees. Australian Breast Cancer Family Study.

Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.

HRAS1 rare minisatellite alleles and breast cancer in Australian women under age forty years.

BACKGROUND: A recent meta-analysis of 23 studies supported the empirically derived hypothesis that women who lack one of the four common minisatellite alleles at the HRAS1 locus are at increased risk of breast cancer. These studies relied on visual sizing of alleles on electrophoretic gels and may have underreported rare alleles. We determined whether this hypothesis applied to early-onset breast cancer by using a new method to size minisatellite alleles. METHODS: We conducted a population-based, case-control-family study of 249 Australian women under 40 years old at diagnosis of a first primary breast cancer and 234 randomly selected women, frequency matched for age. We sized HRAS1 minisatellite alleles with an Applied Biosystems model 373 automated DNA sequencer and GENESCAN(TM) software. All P values are two-sided. RESULTS: We found no association of rare alleles with breast cancer, before or after adjustment for risk factors and irrespective of how their effects were modeled (crude odds ratio = 1.04; 95% confidence interval [CI] = 0.071-1.53; P =.8). The rare allele frequency was 0. 173 (95% CI = 0.149-0.197), three times the pooled estimate of 0.058 (95% CI = 0.050-0.066) from previous studies (P<.001), and was similar for case subjects, 0.177 (95% CI = 0.143-0.221), and control subjects, 0.169 (95% CI = 0.135-0.203) (P =.7). CONCLUSION: There was no support for an association between rare HRAS1 alleles and the risk of early-onset breast cancer, despite 80% power to detect effects of the magnitude of those associations (1.7-fold) previously suggested. IMPLICATIONS: The question of whether cancer risk is associated with rare minisatellite HRAS1 alleles needs to be revisited with the use of new methods that have a greater ability to distinguish rare alleles from similarly sized common alleles.

Population-based estimate of the average age-specific cumulative risk of breast cancer for a defined set of protein-truncating mutations in BRCA1 and BRCA2. Australian Breast Cancer Family Study.

The average breast cancer risk for carriers of a germ-line mutation in BRCA1 or BRCA2 (penetrance) has been estimated from the multiple-case families collected by the Breast Cancer Linkage Consortium (BCLC) to be approximately 80% to age 70. However, women now being tested for these mutations do not necessarily have the intense family history of the BCLC families. Testing for protein-truncating mutations in exons 2, 11, and 20 of BRCA1 and exons 10 and 11 of BRCA2 was conducted in a population-based sample of 388 Australian women with breast cancer diagnosed before age 40. Onset of breast cancer was analyzed in the known and potential mutation-carrying first- and second-degree female relatives of cases found to carry a mutation. Of the 18 mutation-carrying cases (9 BRCA1 and 9 BRCA2), only 5 (1 BRCA1 and 4 BRCA2) had at least one affected relative, so family history of breast cancer was not a strong predictor of mutation status in this setting. The risk in mutation carriers was, on average, 9 times the population risk [95% confidence interval (CI), 4-23; P < 0.001]. Penetrance to age 70 was 40% (95% CI, 15-65%), about half that estimated from BCLC families. By extrapolation, approximately 6% (95% CI, 2-20%) of breast cancer before age 40 may be caused by protein-truncating mutations in BRCA1 or BRCA2. Breast cancer risk in BRCA1 or BRCA2 mutation carriers may be modified by other genetic or environmental factors. Genetic counselors may need to take into account the family history of the consultand.