MSR1 repeats modulate gene expression and affect risk of breast and prostate cancer.

Background: MSR1 repeats are a 36-38 bp minisatellite element that have recently been implicated in the regulation of gene expression, through copy number variation (CNV). Patients and methods: Bioinformatic and experimental methods were used to assess the distribution of MSR1 across the genome, evaluate the regulatory potential of such elements and explore the role of MSR1 elements in cancer, particularly non-familial breast cancer and prostate cancer. Results: MSR1s are predominately located at chromosome 19 and are functionally enriched in regulatory regions of the genome, particularly regions implicated in short-range regulatory activities (H3K27ac, H3K4me1 and H3K4me3). MSR1-regulated genes were found to have specific molecular roles, such as serine-protease activity (P = 4.80 × 10-7) and ion channel activity (P = 2.7 × 10-4). The kallikrein locus was found to contain a large number of MSR1 clusters, and at least six of these showed CNV. An MSR1 cluster was identified within KLK14, with 9 and 11 copies being normal variants. A significant association with the 9-copy allele and non-familial breast cancer was found in two independent populations (P = 0.004; P = 0.03). In the white British population, the minor allele conferred an increased risk of 1.21-3.51 times for all non-familial disease, or 1.7-5.3 times in early-onset disease. The 9-copy allele was also found to be associated with increased risk of prostate cancer in an independent population (odds ratio = 1.27-1.56; P =0.009). Conclusions: MSR1 repeats act as molecular switches that modulate gene expression. It is likely that CNV of MSR1 will affect risk of development of various forms of cancer, including that of breast and prostate. The MSR1 cluster at KLK14 represents the strongest risk factor identified to date in non-familial breast cancer and a significant risk factor for prostate cancer. Analysis of MSR1 genotype will allow development of precise stratification of disease risk and provide a novel target for therapeutic agents.

Delineating the phenotypic spectrum of Bainbridge-Ropers syndrome: 12 new patients with de novo, heterozygous, loss-of-function mutations in ASXL3 and review of published literature.

BACKGROUND: Bainbridge-Ropers syndrome (BRPS) is a recently described developmental disorder caused by de novo truncating mutations in the additional sex combs like 3 (ASXL3) gene. To date, there have been fewer than 10 reported patients. OBJECTIVES: Here, we delineate the BRPS phenotype further by describing a series of 12 previously unreported patients identified by the Deciphering Developmental Disorders study. METHODS: Trio-based exome sequencing was performed on all 12 patients included in this study, which found a de novo truncating mutation in ASXL3. Detailed phenotypic information and patient images were collected and summarised as part of this study. RESULTS: By obtaining genotype:phenotype data, we have been able to demonstrate a second mutation cluster region within ASXL3. This report expands the phenotype of older patients with BRPS; common emerging features include severe intellectual disability (11/12), poor/ absent speech (12/12), autistic traits (9/12), distinct face (arched eyebrows, prominent forehead, high-arched palate, hypertelorism and downslanting palpebral fissures), (9/12), hypotonia (11/12) and significant feeding difficulties (9/12) when young. DISCUSSION: Similarities in the patients reported previously in comparison with this cohort included their distinctive craniofacial features, feeding problems, absent/limited speech and intellectual disability. Shared behavioural phenotypes include autistic traits, hand-flapping, rocking, aggressive behaviour and sleep disturbance. CONCLUSIONS: This series expands the phenotypic spectrum of this severe disorder and highlights its surprisingly high frequency. With the advent of advanced genomic screening, we are likely to identify more variants in this gene presenting with a variable phenotype, which this study will explore.

Life expectancy and death from cardiomyopathy amongst carriers of Duchenne and Becker muscular dystrophy in Scotland.

OBJECTIVES: To assess life expectancy and cardiovascular mortality in carriers of Duchenne and Becker muscular dystrophy. DESIGN: Family pedigrees of individuals affected with these conditions, held by the four genetics centres in Scotland, were examined to identify a cohort of definite carriers. Electronic death registration data, held by the General Register Office for Scotland, were used to identify death certificates of carriers who had died, to obtain age at death and cause of death. Survival and mortality data were obtained for the general population for comparison. PATIENTS: 397 definite carriers in 202 pedigrees were identified from which 94 deaths were identified by record linkage to death certificates. MAIN OUTCOME MEASURES: Observed numbers surviving to certain ages and numbers dying of cardiac causes were compared with expected numbers calculated from general population data. RESULTS: There were no significant differences between observed and expected numbers surviving to ages 40-90. The standardised mortality ratio for the 371 carriers alive in 1974 was 0.53 (95% confidence interval 0.32 to 0.82). CONCLUSIONS: Whereas female carriers may have clinical features of cardiomyopathy, this study does not suggest that this is associated with reduced life expectancy or increased risk of cardiac death. Routine cardiac surveillance of obligate carriers is therefore probably unnecessary.

Evidence for loss of heterozygosity of 5q in sporadic haemangiomas: are somatic mutations involved in haemangioma formation?

BACKGROUND/AIMS: Haemangiomas are common benign tumours of infancy that consist of rapidly proliferating endothelial cells. A locus for an autosomal dominant predisposition to haemangioma has been identified recently on chromosome 5q. This study aimed to investigate loss of heterozygosity on chromosomes 5 and 9 in haemangiomas. METHODS: Sporadic proliferative phase haemangiomas were microdissected. Polymerase chain reaction amplification and analysis of microsatellite markers on chromosomes 5 and 9 was carried out. RESULTS: There was a significant loss of heterozygosity for markers on chromosome 5q in haemangioma tissue, when compared with either markers from chromosome 5p (p < 0.05) or markers from chromosome 9 (p < 0.05). CONCLUSIONS: These results suggest that haemangioma formation might be associated with somatic mutational events, and provides evidence that a locus on 5q is involved in the formation of sporadic haemangiomas.

Two common endoglin mutations in families with hereditary hemorrhagic telangiectasia in the Netherlands Antilles: evidence for a founder effect.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant bleeding disorder characterized by localized angiodysplasia. Mutations in either of two genes, endoglin or ALK-1, can cause HHT. Both genes encode putative receptors for the transforming growth factor-beta superfamily of ligands. Many mutations in each gene have been identified in HHT kindreds from around the world, and with few exceptions mutations are unique and family specific. The prevalence of HHT in the Leeward Islands of the Netherlands Antilles is possibly the highest of any geographical location. We wished to establish whether this high prevalence is due to a genetic founder effect or to multiple mutational events. HHT kindreds from the Netherlands Antilles and The Netherlands were screened for mutations in the two genes associated with HHT. Haplotype analysis of a 5-cM region on chromosome 9 flanking the endoglin gene revealed three distinct disease haplotypes in the ten Antillean families studied. Seven of these families share a splice-site mutation in exon 1 of endoglin. Two other Antillean families share a missense mutation in exon 9a of endoglin. This mutation was also found in a Dutch family that shares the same disease haplotype as the Antillean families with this mutation. Thus it appears that HHT in the Netherlands Antilles is due to a limited number of ancestral mutations in the endoglin gene, and that one of these mutations was introduced into the African slave population by a Dutch colonist. The limited scope of mutations suggests that a presymptomatic screening program for HHT would be feasible in this population.

Allelic and locus heterogeneity in inherited venous malformations.

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-beta binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor-complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT.

The activin receptor-like kinase 1 gene: genomic structure and mutations in hereditary hemorrhagic telangiectasia type 2.

The activin receptor-like kinase 1 gene (ALK-1) is the second locus for the autosomal dominant vascular disease hereditary hemorrhagic telangiectasia (HHT). In this paper we present the genomic structure of the ALK-1 gene, a type I serine-threonine kinase receptor expressed predominantly in endothelial cells. The coding region is contained within nine exons, spanning < 15 kb of genomic DNA. All introns follow the GT-AG rule, except for intron 6, which has a TAG/gcaag 5' splice junction. The positions of introns in the intracellular domain are almost identical to those of the mouse serine-threonine kinase receptor TSK-7L. By sequencing ALK-1 from genomic DNA, mutations were found in six of six families with HHT either shown to link to chromosome 12q13 or in which linkage of HHT to chromosome 9q33 had been excluded. Mutations were also found in three of six patients from families in which available linkage data were insufficient to allow certainty with regard to the locus involved. The high rate of detection of mutations by genomic sequencing of ALK-1 suggests that this will be a useful diagnostic test for HHT2, particularly where preliminary linkage to chromosome 12q13 can be established. In two cases in which premature termination codons were found in genomic DNA, the mutant mRNA was either not present or present at barely detectable levels. These data suggest that mutations in ALK-1 are functionally null alleles.

Dietary fish oil enhances circulating interferon-gamma in mice during listeriosis without altering in vitro production of this cytokine.

The objective of this study was to investigate the impact of feeding mice a diet rich in n-3 polyunsaturated fatty acids (PUFA) from fish oil on the interferon-gamma (IFN-gamma) response during an active infection with Listeria monocytogenes. Weanling female C3H/Hen mice were fed experimental diets containing 20% by weight one of the following fats: soybean oil, lard, or a mixture of menhaden fish oil and corn oil (17:3, w/w). After 4 weeks, mice were injected with 10(5) live L. monocytogenes, and the concentration of IFN-gamma in serum and spleen was determined 0, 2, 4, and 7 days postinfection by enzyme-linked immunosorbent assay (ELISA). Fish oil-fed mice showed significantly higher IFN-gamma in their blood at 2 and 4 days postchallenge compared with mice fed the soybean oil-containing or lard-containing diets (p < 0.001). A higher concentration of IFN-gamma was also found in the spleen homogenate of fish oil-fed mice on day 4 postchallenge (p < 0.005). To examine in vitro IFN-gamma production, splenocytes were isolated from fish oil-fed and soybean oil-fed mice on day 4 postchallenge and cultured with concanavalin A (1 microgram/ml and 10 micrograms/ml) for 24 and 48 h. There were no significant differences in the IFN-gamma concentration in cell culture supernatants between these diet treatments. This study demonstrated that the elevation in the concentration of IFN-gamma in blood and spleen during murine listeriosis is accentuated and prolonged by dietary n-3 PUFA, and these effects may not be due to changes in IFN-gamma production.

Dietary fish oil reduces survival and impairs bacterial clearance in C3H/Hen mice challenged with Listeria monocytogenes.

1. To investigate the effect of dietary fat source on host resistance to intracellular pathogens, weanling female C3H/Hen mice were fed one of three experimental diets containing, 20% by weight, lard, soybean oil or 17% menhaden fish oil plus 3% corn oil. After 4 weeks, survival of mice (n = 12/treatment group) injected intraperitoneally with 2 x 10(6) colony forming units of live Listeria monocytogenes was determined. In a second study, bacterial clearance from the liver and spleen at 2, 4 and 7 days post-challenge was determined (n = 8/treatment group). 2. We found that the survival of mice fed the diets with soybean oil or menhaden fish oil was significantly lower than those fed lard (P < 0.05). Survival rates were 58% (7/12), 33% (4/12) and 100% (12/12), respectively, for mice fed soybean oil, menhaden fish oil and lard. In the second study, mice fed menhaden fish oil had approximately 1 log10 greater bacteria in their spleens at day 4 than mice fed lard or soybean oil (P < 0.001). There were no significant treatment differences in the number of bacteria recovered from liver samples. 3. In summary, dietary fat source significantly affects murine resistance to Listeria, with diets rich in n-3 polyunsaturated fatty acids, such as from fish oil, having the most detrimental effect.

Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2.

Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.

Clinical heterogeneity in hereditary haemorrhagic telangiectasia: are pulmonary arteriovenous malformations more common in families linked to endoglin?

Pulmonary arteriovenous malformations (PAVMs) occur in up to 27% of patients with hereditary haemorrhagic telangiectasia (HHT) and are associated with a rate of paradoxical cerebral embolism at presentation of up to 36%. At least two different loci have been shown for HHT. Mutations in endoglin have been found in some families and the locus designated ORW1. In other families this locus has been excluded. In this paper we confirm that in families linked to ORW1 there is a prevalence of PAVMs among affected members of 29.2%, compared to a prevalence of 2.9% in families in which this locus has been excluded (chi 2 = 19.2, p < 0.001). This information can be used to decide how to screen HHT patients for PAVMs.

A second locus for hereditary hemorrhagic telangiectasia maps to chromosome 12.

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Rendu-Weber (ORW) disease is an autosomal dominant vascular dysplasia. Initial linkage studies identified an ORW gene localized to 9q33-q34 but with some families clearly excluding this region. A probable correlation in clinical phenotype between the 9q3-linked families and unlinked families was described with a significantly lower incidence of pulmonary arteriovenous malformations observed in the unlinked families. In this study we examined four unrelated ORW families for which linkage to chromosome 9q33-q34 has been previously excluded. Linkage was established for all four families to markers on chromosome 12, with a combined maximum lod score of 10.77 (theta = 0.04) with D12S339. Mapping of crossovers using haplotype analysis indicated that the candidate region lies in an 11-CM interval between D12S345 and D12S339, in the pericentromeric region of chromosome 12. A map location for a second ORW locus is thus established that exhibits a significantly reduced incidence of pulmonary involvement.

Effects of three occlusive dressing materials on healing of full-thickness skin wounds in dogs.

The effects of 3 occlusive dressing materials and a standard, nonadherent dressing material on healing of full-thickness skin defects were evaluated in dogs. Two wounds measuring 2 x 2 cm were created bilaterally (4 wounds/dog) on the dorsolateral aspect of the trunk of 12 Beagles. Wound treatments were evenly distributed between 4 sites, using a Latin square design. Treatments evaluated were: equine amnion (group A), biosynthetic hydrogel dressing (group B), transparent polyethylene sheeting (group T), and a semi-occlusive rayon/polyethylene, nonadherent dressing (group C). Rates of contraction and epithelialization of group-A wounds were significantly greater than those of wounds of groups C, B, and T. On days 14, 21, and 28, mean percentage of wound contraction and mean percentage of total wound healed in group A exceeded those wounds in groups C, B, and T. On day 28, wounds in group A were significantly smaller than wounds in groups B and T, but were not significantly smaller than wounds in group C. All wounds in group A achieved 100% healing during the 28-day study period. Mean time for complete healing of group-A wounds was 21 days. The percentages of wounds completely healed by day 28 for groups B, C, and T were 25, 67, and 25%, respectively. Results indicate that use of equine amnion as an occlusive biological dressing on full-thickness wounds in dogs increases rate of healing.

Evaluation of a one-step surgical preparation technique in dogs.

The efficacy of a 1-step surgical preparation technique for skin of dogs prior to elective ovariohysterectomy was evaluated. Dogs randomly assigned to group 1 (n = 30) had their skin prepared for surgery by use of a 2-step method, whereas the skin of dogs in group 2 (n = 30) was prepared for surgery by use of a commercially available product for a 1-step technique. Culture plates for quantitative bacterial counts were applied to the proposed incision site on dogs under general anesthesia after hair at the site was clipped and vacuumed but before antiseptic was applied. A second quantitative bacterial culture plate was applied to the proposed incision site after completion of the surgical preparation technique. Surgeries were routinely completed, and dogs were evaluated by physical examination the next day and at the time of suture removal (7 to 10 days after surgery) for complications. Postoperative complications were minor and consisted primarily of subcutaneous swelling, which resolved with time. All cultures obtained prior to skin preparation included bacteria or yeast. Sixteen cultures obtained after skin preparation (group 1, n = 11; group 2, n = 5) included bacteria or yeast. The total number of colonies of potential pathogens (Staphylococcus sp and Enterobacteriaceae) on the preparation cultures was 9,339; 4 colonies were counted on the postpreparation cultures. Potential bacterial pathogens, ie Streptococcus intermedius and gram-negative bacteria, were isolated from dogs prepared with the 2-step technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure and molecular characterization of Fusobacterium necrophorum biovars.

The ultrastructural features and molecular components of 18 strains of Fusobacterium necrophorum biovars A, AB and B, isolated from animal and human infections, were examined by electron microscopy, multilocus enzyme electrophoresis (MEE) and by sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-PAGE). High resolution scanning electron microscopy revealed that the strains possessed a convoluted surface pattern. Transmission electron microscopy showed that all strains possessed a cell wall structure typical of gram-negative bacteria. Bleb formation was not uncommon. Numerous extracellular materials, resembling lipopolysaccharide (LPS) fragments, surrounded cells of both human strains and biovar B animal strains. Biovar A field strains revealed capsules as stained by ruthenium red whereas a stock culture strain showed the capsule only when immunostabilized with hyperimmune serum. Starch gel electrophoresis showed all strains to possess adenyl kinase, glutamate dehydrogenases and lactate dehydrogenase; each enzyme migrated uniformly (monomorphic) among the strains and represented an electrotype. However, SDS-PAGE indicated differences in the protein profiles between all of the strains; the most distinctly different was a human isolate (FN 606). Silver staining to detect LPS showed extensive "ladder" patterns among the majority of biovar A strains but not in the animal biovar B strains. Immunoblotting of LPS with a rabbit antiserum prepared against phenol extracted LPS from a biovar A animal isolate (LA 19) suggested marked variability in the LPS antigens among the isolates studied.

Release of tumor necrosis factor-alpha from bovine alveolar macrophages stimulated with bovine respiratory viruses and bacterial endotoxins.

The release of tumor necrosis factor-alpha (TNF-alpha) from cultured bovine alveolar macrophages (BAM) was evaluated following stimulation of BAM with bovine herpesvirus-1 (BHV-1), parainfluenza-3 (PI-3) virus, bovine respiratory syncytial virus (BRSV), Escherichia coli 0111:B4 endotoxin, Pasteurella haemolytica type 1 endotoxin, Pasteurella multocida endotoxin, and virus/endotoxin combinations. A cytotoxic assay system using Georgia bovine kidney cells as targets was used to measure TNF-alpha activity. The cytotoxic activity was neutralized by an anti-human TNF-alpha monoclonal antibody. Stimulation of BAM with 1 median tissue culture infectious dose (TCID50) of live or ultraviolet (UV)-inactivated PI-3 virus/cell resulted in release of TNF-alpha in significantly (P less than 0.05) higher amounts than sham-induced BAM. The quantities of TNF-alpha released after live or UV-inactivated BHV-1 or BRSV induction were not significantly higher than sham-induced BAM. E. coli 0111:B4, P. haemolytica type 1 and P. multocida endotoxins stimulated TNF-alpha release in a dose-dependent manner. Sequential exposure of BAM to 1 TCID50 per cell of either live BHV-1, PI-3 virus or BRSV and then 5 micrograms ml-1 of either E. coli 0111:B4, P. haemolytica type 1 or P. multocida endotoxin caused a significant (P less than 0.05) reduction in detectable TNF-alpha in seven of nine virus/endotoxin combinations tested, when compared with 5 micrograms ml-1 of endotoxin alone. Parainfluenza-3 virus/endotoxin combinations stimulated higher TNF-alpha release when compared with other virus/endotoxin combinations. Five out of six test animals had serum-neutralizing antibodies to PI-3 virus, one out of six had serum-neutralizing antibodies to BHV-1, and two out of six had serum-neutralizing antibodies to BRSV, suggesting a possible relationship between serum neutralizing antibodies and TNF-alpha release from in vitro cultivated BAM.

Serum distribution of iodine after oral administration of ethylenediamine dihydriodide in cattle.

Serum concentrations of iodine were determined after cattle were given ethylenediamine dihydriodide (EDDI) orally at dosages ranging from 0.0 (placebo) to 0.77 mg/kg of body weight/day. The serum iodine concentration was correlated with the dosage of EDDI. A rate of 0.11 mg EDDI/kg/day was correlated with serum iodine concentrations (20 to 80 micrograms/dl) previously found to be effective in preventing foot rot in cattle. A linear dose-response curve that was generated could be helpful in predicting dosage of EDDI if the serum iodine concentration is known.