Hereditary breast cancer: syndromes, tumour pathology and molecular testing.

Hereditary factors account for a significant proportion of breast cancer risk. Approximately 20% of hereditary breast cancers are attributable to pathogenic variants in the highly penetrant BRCA1 and BRCA2 genes. A proportion of the genetic risk is also explained by pathogenic variants in other breast cancer susceptibility genes, including ATM, CHEK2, PALB2, RAD51C, RAD51D and BARD1, as well as genes associated with breast cancer predisposition syndromes - TP53 (Li-Fraumeni syndrome), PTEN (Cowden syndrome), CDH1 (hereditary diffuse gastric cancer), STK11 (Peutz-Jeghers syndrome) and NF1 (neurofibromatosis type 1). Polygenic risk, the cumulative risk from carrying multiple low-penetrance breast cancer susceptibility alleles, is also a well-recognised contributor to risk. This review provides an overview of the established breast cancer susceptibility genes as well as breast cancer predisposition syndromes, highlights distinct genotype-phenotype correlations associated with germline mutation status and discusses molecular testing and therapeutic implications in the context of hereditary breast cancer.

Ultraviolet radiation exposure to the face in patients with xeroderma pigmentosum and healthy controls: applying a novel methodology to define photoprotection behaviour.

BACKGROUND: In xeroderma pigmentosum (XP), the main means of preventing skin and eye cancers is extreme protection against ultraviolet radiation (UVR). Protection is most important for the face. OBJECTIVES: We aimed to assess how well patients with XP adhere to medical advice to protect against UVR by objectively estimating the mean daily dose of UVR to the face. METHODS: We objectively estimated the UVR dose to the face in 36 patients with XP and 25 healthy individuals over 3 weeks in the summer. We used a new methodology which combined UVR dose measurements from a wrist-worn dosimeter with an activity diary record of face photoprotection behaviour for each 15-min period spent outside. A protection factor was associated with each behaviour, and the data were analysed using a negative binomial mixed-effects model. RESULTS: The mean daily UVR dose (weighted for DNA damage capacity) to the face in the patients with XP was 0·13 standard erythemal doses (SEDs) (mean in healthy individuals = 0·51 SED). There was wide variation between patients (range < 0·01-0·48 SED/day). Self-caring adult patients had a very similar UVR dose to the face as cared-for patients (0·13 vs. 0·12 SED/day), despite photoprotecting much more poorly when outside, because the self-caring adults were outside in daylight much less. CONCLUSIONS: Photoprotection behaviour varies widely within the XP group indicating that nonadherence to photoprotection advice is a significant issue. The timing and duration of going outside are as important as photoprotective measures taken when outside, to determine the UVR exposure to the face. This new methodology will be of value in identifying the sources of UVR exposure in other conditions in which facial UVR exposure is a key outcome, particularly in patients with multiple nonmelanoma skin cancers.

Population Viability Analysis of the Endangered Roan Antelope in Ruma National Park, Kenya, and Implications for Management.

Population viability analysis (PVA) was used to (1) establish causes of roan population decline for the past 30 years in Ruma National Park (RNP), the only park where wild roans remain in Kenya, and (2) predict the probability of roan persistence under existing and alternative management options. PVA was done using long-term data based on population dynamics, life history, climatic conditions, and expert knowledge. Poaching was identified as the main cause of roan decline in RNP. Several antipoaching and prioritized habitat management interventions to promote population recovery and sustainable conservation of roans are described. PVA predictions indicated that, without these interventions, the roan population cannot persist more than 3 decades. Furthermore, ensuring sustainable conservation of roans in RNP will boost tourism in Western Kenyan and thus alleviate poverty in this part of the country. Improved income from tourism will reduce the possible pressures from hunting and give greater incentives for local people to be actively engaged in roan conservation.

Identification of olfactory receptor genes in Atlantic salmon Salmo salar.

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. The key components of the molecular pathways involved in imprinting and homing, however, are still unknown. Aquatic chemical cues are received through the nares and into the nasal cavity that contains a single olfactory organ, the olfactory rosette. The olfactory rosette contains sensory neurons, each of which is thought to express only one olfactory receptor. If odorants are involved in salmonid homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, to understand the molecular basis for imprinting and homing in Atlantic salmon Salmo salar it is important to identify and characterize the repertoire of olfactory receptors in this species. The first public assembly of the S. salar genome was searched for genes encoding three of the superfamilies of fish olfactory receptors: V2R-like (olfc), V1R-like (ora) and main olfactory receptor (mor). A further six ora genes were added to ora1 and ora2, which had been described previously. In addition, 48 putative mors were identified, 24 of which appear to be functional based on their gene structures and predicted amino-acid sequences. Phylogenetic analyses were then used to compare these S. salar olfactory receptor genes with those of zebrafish Danio rerio, two pufferfish species Takifugu rubripes and Tetraodon nigroviridis, medaka Oryzias latipes and three-spined stickleback Gasterosteus aculeatus.

Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar).

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.

Novel monogenic diabetes mutations in the P2 promoter of the HNF4A gene are associated with impaired function in vitro.

AIMS: Mutations in HNF4A cause a form of monogenic beta-cell diabetes. We aimed to identify mutations in the pancreas-specific P2 promoter of HNF4A in families with suspected HNF4A diabetes and to show that they impaired the function of the promoter in vitro. METHODS: We screened families with a clinical suspicion of HNF4A monogenic beta-cell diabetes for mutations in the HNF4A P2 promoter. We investigated the function of the previously reported HNF4A P2 promoter mutation -192C>G linked to late-onset diabetes in several families, along with two new segregating mutations, in vitro using a modified luciferase reporter assay system with enhanced sensitivity. RESULTS: We identified two novel HNF4A P2 promoter mutations that co-segregate with diabetes in two families, -136A>G and -169C>T. Both families displayed phenotypes typical of HNF4A monogenic beta-cell diabetes, including at least two affected generations, good response to sulphonylurea treatment and increased birthweight and/or neonatal hypoglycaemia. We show that both of these novel mutations and -192C>G impair the function of the promoter in transient transfection assays. CONCLUSIONS: Two novel mutations identified here and the previously identified late-onset diabetes mutation, -192C>G, impair the function of the HNF4A P2 promoter in vitro.

The PG500 series: novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy.

Heparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

Can the initial success of the Malawi ART scale-up programme be sustained? The example of Queen Elizabeth Central Hospital, Blantyre.

The antiretroviral therapy clinic of Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi was established as a fee-paying clinic in 2000. In 2004 a successful transition to free-of-charge antiretroviral therapy (ART) provision was made with the introduction of the national ART scale-up programme. Despite the human resource crisis in the healthcare system, remarkable improvements in quantity and quality of care, a reduction of defaulters, favourable ART outcomes and better access to ART for the poor, women and children were achieved. A number of challenges need to be overcome to sustain the initial success of the national ART scale-up programme in QECH, the most important being the shortage of ART staff in relation to the ever-expanding patient population.

Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus and Chlamys islandica.

Characterization of DNA that remains in seafood products after skin, scales, and shells are removed is widely used in forensic species identification, however, ordinary methods may be prohibitively expensive or time-consuming if large sample series need to be discriminated. Forensic discrimination of two species of bivalves commercially harvested from the North Atlantic, sea scallops (Placopecten magellanicus) and Icelandic scallops (Chlamys islandica), was made by means of species-specific oligonucleotides (SSOs) in a multiplex polymerase chain reaction (PCR). The test is a simultaneous in vitro amplification of a portion of the mitochondrial Cytochrome Oxidase I locus with a PCR anchor primer for a sequence identical in both species, and two alternative SSOs that selectively amplify either a 619-bp in Placopecten or a 459-bp DNA fragment in Chlamys. Fragment size and thus species identity are determined directly by gel electrophoresis. In the forensic application, analysis of more than 900 scallops from a series of samples seized from two fishing vessels showed significantly variable proportions of the species from the closed and open fisheries (Placopecten versus Chlamys, respectively). The multiplex SSO test provides a direct means of forensic identification of large population sample series, without the necessity of secondary DNA sequencing, RFLP mapping, or fingerprinting, and can be adapted to other loci and species.

HMG CoA reductase-inhibitor-related myopathy and the influence of drug interactions.

We report four cases of rhabdomyolysis and severe, disabling myopathy associated with HMG CoA reductase-inhibitor therapy. Patient developed symptoms following the addition of roxithromycin to combination lipid-lowering therapy with simvastatin and gemfibrozil. Patients 2 and 3 became symptomatic after developing acute on chronic renal impairment while taking simvastatin. The muscle biopsy of patient 3 revealed a necrotizing myopathy and the presence of inclusion bodies. Patient 4 developed symptoms within 4 weeks of starting cerivastatin monotherapy. The four cases illustrate the importance of considering the potential for drug interactions and making appropriate dosage adjustments for renal insufficiency in patients receiving HMG CoA reductase therapy.

How to tell a sea monster: molecular discrimination of large marine animals of the North Atlantic.

Remains of large marine animals that wash onshore can be difficult to identify due to decomposition and loss of external body parts, and in consequence may be dubbed "sea monsters." DNA that survives in such carcasses can provide a basis of identification. One such creature washed ashore at St. Bernard's, Fortune Bay, Newfoundland, in August 2001. DNA was extracted from the carcass and enzymatically amplified by the polymerase chain reaction (PCR): the mitochondrial NADH2 DNA sequence was identified as that of a sperm whale (Physeter catodon). Amplification and sequencing of cryptozoological DNA with "universal" PCR primers with broad specificity to vertebrate taxa and comparison with species in the GenBank taxonomic database is an effective means of discriminating otherwise unidentifiable large marine creatures.

Analysis of the role of recA in phenotypic switching of Pseudomonas tolaasii.

Switching between the pathogenic smooth (1116S) and nonpathogenic rough (1116R) forms of Pseudomonas tolaasii occurs due to the reversible duplication of a 661-bp element within the pheN locus. Disruption of the chromosomal recA locus of 1116S and 1116R produced strains 1116SrecA and 1116RrecA, respectively, which showed typical loss of UV resistance. Switching from the smooth to the rough form was virtually eliminated in the 1116SrecA strain, whereas the extent of switching from the rough to the smooth form was almost identical in 1116R and 1116RrecA. It is concluded that phenotypic switching from 1116S to 1116R is recA dependent whereas that from 1116R to 1116S is recA independent.

Psychiatrists' recommendations for improving bicultural training and Maori mental health services: a New Zealand survey.

OBJECTIVE: In the context of Maori being over-represented as clients, and underrepresented as professionals in New Zealand's mental health system, this study ascertained the beliefs of New Zealand's psychiatrists about issues pertaining to Maori mental health. The overriding objective was to gather recommendations as to how to improve bicultural training and mental health services for Maori. METHOD: A questionnaire involving closed and open-ended questions was sent to 335 New Zealand psychiatrists. RESULTS: Of the 247 psychiatrists (74%) responding, 40% believed their training had prepared them to work effectively with Maori. Recommendations for improving training focused on the need for greater understanding of Maori perspectives of well-being. Recommendations for improving mental health services for Maori highlighted the need for more Maori professionals and for Maori-run services. No psychiatrists thought that pakeha clinicians should not work with Maori clients, but the majority (70%) recognised the need to consult with Maori staff when doing so. Twenty-eight psychiatrists (11.3%), all male, New Zealand born, and with 10 or more years clinical experience, believed that Maori were biologically or genetically more predisposed than others to mental illness. Several respondents offered other racist comments. CONCLUSIONS: The high response rate and the many positive recommendations suggest a high level of constructive interest in these issues among psychiatrists. Comparisons with a simultaneous survey of psychologists are made. It is hoped that the recommendations might inform those responsible for training programs and for providing or purchasing mental health services.

Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms.

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non-pathogenic form (designated 1116R). This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins. Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb Xhol-BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R. In order to characterize the genetic basis of this variation, the pheN locus (designated pheN') was cloned from 1116R and its nucleotide sequence determined. A 661 bp duplication was identified within pheN' introducing a frameshift mutation in the predicted pheN open reading frame (ORF). A resulting predicted ORF of pheN' designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain. Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form. Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S. Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form. The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99kDa and 77kDa cross-reactive bands in 1116S and 1116R respectively. It is concluded that the spontaneous colony-sectoring event that results in the 1116R phenotypic variant form of P. tolaasii arises owing to a 661 bp DNA duplication within the 5' end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.

Identification of a novel transcript disrupted by a balanced translocation associated with DiGeorge syndrome.

Most cases of DiGeorge syndrome (DGS) and related abnormalities are associated with deletions within 22q11. Shortest region of deletion overlap (SRO) mapping previously identified a critical region (the DGCR) of 500 kb, which was presumed to contain a gene or genes of major effect in the haploinsufficiency syndromes. The DGCR also contains sequences disrupted by a balanced translocation that is associated with DGS--the ADU breakpoint. We have cloned sequences at the breakpoint and screened for novel genes in its vicinity. A series of alternatively spliced transcripts expressed during human and murine embryogenesis, but with no obvious protein encoding potential, were identified. The gene encoding these RNAs has been named DGCR5 and it is disrupted by the patient ADU breakpoint. DGCR5 is distinct from the DGCR3 open reading frame (ORF) previously shown to be interrupted by the ADU translocation, although DGCR3 is embedded within a DGCR5 intron and in the same (predicted) transcriptional orientation. No mutations of DGCR5 have yet been detected. By analogy to other loci encoding conserved, nontranslated RNAs, it is possible that DGCR5 originates from a cis-acting transcriptional control element in the vicinity of the ADU/VDU breakpoint. Disruption of such an element would result in altered transcription of neighboring genes secondary to a position effect, a hypothesis in keeping with recent refinement of the SRO placing the ADU breakpoint outside the DGCR.

Characterisation of a short interspersed repeat (Mermaid) that has family members on human chromosome 21 and elsewhere in the human genome.

To understand the architecture of the human genome, we need a complete definition of all the repeat sequence families, as these make up the majority of human DNA. We have isolated a small DNA fragment from human chromosome 21 and have used sequence analysis of this fragment to uncover a new low copy repeat element of approximately 300 bp that we term the Mermaid repeat. This repeat is related to, but is different from, the MER12 repeat and is interspersed in the genome. Mermaid family members that we have studied are between 81%-87% identical to our preliminary consensus sequence. Therefore, we have added a new member to the large collection of human repetitive elements. In addition, we have mapped a Mermaid repeat to a telomeric position on the long arm of human chromosome 21, at 21q22.3.

Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus.

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii.

Cloning and mapping of murine Nfe2l1.

The murine homologue of the human NFE2L1 basic leucine-zipper gene was isolated from an early embryo library. The deduced amino acid sequence shows 97% identity between the two proteins. Significant sequence similarity is also seen with the p45 subunit of NF-E2 and with the Drosophila CNC protein. Murine Nfe2l1 maps to chromosome 11DE with similar sequence at 7D1-7F1 and 2E4-2G.

Tellurium and Selenium Resistance in Rhizobia and Its Potential Use for Direct Isolation of Rhizobium meliloti from Soil.

Forty-eight Rhizobium and Bradyrhizobium strains were screened for resistance to tellurite, selenite, and selenate. High levels of resistance to the metals were observed only in Rhizobium meliloti and Rhizobium fredii strains; the MICs were 2 to 8 mM for Te(IV), >200 mM for Se(VI), and 50 to 100 mM for Se(IV). Incorporation of Se and Te into growth media permitted us to directly isolate R. meliloti strains from soil. Mutant strains of rhizobia having decreased levels of Se and Te resistance were constructed by Tn5 mutagenesis and were found to have transposon insertions in DNA fragments of different sizes. Genomic DNAs from Te rhizobium strains failed to hybridize with Te determinants from plasmids RP4, pHH1508a, and pMER610.