Paul K. Lovelock

Genetic and Histopathologic Evaluation of BRCA1 and BRCA2 DNA Sequence Variants of Unknown Clinical Significance

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one‐step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with ‘null’ mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR‐related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement (‘null’) and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild‐type CFTR activity. The long‐term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype‐phenotype correlations.

Familial Paget's disease of bone: nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree.

Pagets disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Pagets disease but the molecular basis remains largely unknown. Susceptibility loci for Pagets disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q21.1‐q22 (PDB2) in different pedigrees. We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Pagets disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log10 of the odds ratio (LOD) scores < −2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < −2.0) centered on D18S42. These data confirm the genetic heterogeneity of Pagets disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Pagets disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.

Variation in the RAD51 gene and familial breast cancer

IntroductionHuman RAD51 is a homologue of the Escherichia coli RecA protein and is known to function in recombinational repair of double-stranded DNA breaks. Mutations in the lower eukaryotic homologues of RAD51 result in a deficiency in the repair of double-stranded DNA breaks. Loss of RAD51 function would therefore be expected to result in an elevated mutation rate, leading to accumulation of DNA damage and, hence, to increased cancer risk. RAD51 interacts directly or indirectly with a number of proteins implicated in breast cancer, such as BRCA1 and BRCA2. Similar to BRCA1 mice, RAD51-/- mice are embryonic lethal. The RAD51 gene region has been shown to exhibit loss of heterozygosity in breast tumours, and deregulated RAD51 expression in breast cancer patients has also been reported. Few studies have investigated the role of coding region variation in the RAD51 gene in familial breast cancer, with only one coding region variant – exon 6 c.449G>A (p.R150Q) – reported to date.MethodsAll nine coding exons of the RAD51 gene were analysed for variation in 46 well-characterised, BRCA1/2-negative breast cancer families using denaturing high-performance liquid chromatography. Genotyping of the exon 6 p.R150Q variant was performed in an additional 66 families. Additionally, lymphoblastoid cell lines from breast cancer patients were subjected to single nucleotide primer extension analysis to assess RAD51 expression.ResultsNo coding region variation was found, and all intronic variation detected was either found in unaffected controls or was unlikely to have functional consequences. Single nucleotide primer extension analysis did not reveal any allele-specific changes in RAD51 expression in all lymphoblastoid cell lines tested.ConclusionOur study indicates that RAD51 is not a major familial breast cancer predisposition gene.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability

Background and purposeAssays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting.Materials and methodsPost-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls.ResultsPost-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status.ConclusionGiven the significant degree of phenotypic heterogeneity of LCLs after γ-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion, 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

The cystic fibrosis transmembrane conductance regulator (CFTR) regulates the sensitivity of macrophages to bacterial lipopolysaccharide

Cystic fibrosis is a chronic inflammatory disease. Most patients are affected by chronic obstructive lung disease associated with novel patterns of bacterial infection, particularly with Pseudomonas aeruginosa. Amongst the symptoms in CF patients is elevated serum levels of cystic fibrosis antigen, a complex of two S-100 proteins (S100A8 and S100A9; also commonly called MRP8 and MRP14) that are abundant in neutrophils and a subset of macrophages. The S100A8 and S100A9 proteins are found in the sera of both patients and heterozygous asymptomatic individuals. The observation that these proteins are also found expressed in the sera of patients with non-CF lung disease has been used to argue that their expression is a sequel to inflammation.1 S100A8 mRNA has been shown elsewhere to be inducible in macrophages by bacterial lipopolysaccharide (LPS).2 To determine whether CF antigen (S100A8/A9) could be induced in a response to infection in the lung, we examined the level of mRNA after intravenous injection of LPS. In control animals, S100A8 mRNA was almost undetectable in any tissue. Within 2 h of LPS injection, the level of mRNA was massively induced and was easily detected using total RNA from lung. Induction was maintained after 8 h. To dissect the sites of expression, we performed mRNA in situ hybridisation and immunocytochemistry. The former approach confirmed that S100A8 mRNA is widely expressed in the LPS-stimulated lung on a subset of cells with epithelial morphology lining alveoli, but is excluded from the bronchial epithelium. Immunolocalisation using anti-S100A8 antibody gave a different pattern, the only cells expressing high levels of S100A8 antigen had a location, morphology, and abundance consistent with their identity as interstitial and alveolar macrophages. The lack of correlation between mRNA and protein has been observed previously in studies of the trophoblasts (unpublished) and suggests that if the S100A8 protein is produced it may be rapidly secreted or in a complex that cannot be detected with the antibody. The chronic pathology observed in CF has been attributed, in part, to inappropriate hyper-responsiveness to lung infection. We have produced a mouse transgenic line with the G551D mutation,3 which has a somewhat less severe gastrointestinal phenotype than the more common human mutation ∆F508 or a complete null. We, therefore, compared the expression of S100A8 mRNA in CF G551D animals and wild-type litter-mates. The animals used in this study were specific pathogen-free and had no overt lung pathology. The majority of the CF animals had readily detectable S100A8 mRNA in the lung, whereas no such signal was detectable in any of the control animals. We injected groups of control and CF animals with LPS. Unlike the controls, which were not obviously affected by the treatment during the time course of the experiment, the CF animals displayed overt symptoms of endotoxaemia, hunched appearance, raised hair, sweating, and shivering. The level of S100A8 mRNA in the lungs of the LPS-treated CF animals was consistently substantially higher than in any of the control animals. Reprobing of these blots for expression of S100A9 revealed that it was similarly induced by LPS, and expressed at higher levels in unstimulated and LPStreated lungs from G551D CF mice. Hybridisation in situ of the S100A8 mRNA in the CF lungs revealed a much more extensive distribution than in wild type litter mates suggesting that, in part, the effect of the mutation is to increase the number of epithelial cells expressing the mRNA. The number of macrophage-like cells detected by immunolocalisation of the S100A8 protein was also increased; and increased expression per cell is not precluded because the labelling is not quantitative. The major cell population that initiates LPS-mediated pathology is the macrophage, which expresses the receptor

Pharmacological Strategies for the Treatment of the Basic Defect in Cystic Fibrosis

Cystic fibrosis (CF), a common, lethal inherited disease of Caucasians, is a systemic exocrine disorder associated with a dysfunctional chloride channel that restricts or prevents the movement of chloride ions across the apical membrane of epithelial cells [1]. This results in elevated sodium and chloride levels in sweat and airway surface fluid, and in the abnormal composition and hydration of mucus [1–4]. Clinical presentations of the disease are widely heterogeneous, but patients commonly show a predisposition to chronic and fatal lung colonisation by bacterial pathogens such as P. aeruginosa and S. aureus [5]. Although there are important gastroenterological manifestations contributing to morbidity and mortality, the lung involvement is the primary cause of 95% of the mortality [6]. Infants who die from meconium ileus shortly after birth do not have macroscopic lung disease although histological abnormalities can be detected within a few days of life [7]. Lung disease develops over the first few years of life and leads to death, on average in the third decade, from chronic suppurative lung disease [8]. Since the CF gene was discovered in 1989 [9, 10], enormous progress has been made in the understanding of the basic defect and the changes that occur in CF. However, there is still much debate about how different genotypes, infection and inflammatory responses interact, and the exact process by which the genetically determined defect leads to extensive damage and irreversible failure of the lungs is not yet clear.