Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype?

Risk of cutaneous malignant melanoma (CMM) is increased in sun-exposed whites, particularly those with a pale complexion. This study was designed to investigate the relationship of the melanocortin-1 receptor (MC1R) genotype to CMM risk, controlled for pigmentation phenotype. We report the occurrence of five common MC1R variants in an Australian population-based sample of 460 individuals with familial and sporadic CMM and 399 control individuals-and their relationship to such other risk factors as skin, hair, and eye color; freckling; and nevus count. There was a strong relationship between MC1R variants and hair color and skin type. Moreover, MC1R variants were found in 72% of the individuals with CMM, whereas only 56% of the control individuals carried at least one variant (P<.001), a finding independent of strength of family history of melanoma. Three active alleles (Arg151Cys, Arg160Trp, and Asp294His), previously associated with red hair, doubled CMM risk for each additional allele carried (odds ratio 2.0; 95% confidence interval 1. 6-2.6). No such independent association could be demonstrated with the Val60Leu and Asp84Glu variants. Among pale-skinned individuals alone, this association between CMM and MC1R variants was absent, but it persisted among those reporting a medium or olive/dark complexion. We conclude that the effect that MC1R variant alleles have on CMM is partly mediated via determination of pigmentation phenotype and that these alleles may also negate the protection normally afforded by darker skin coloring in some members of this white population.     

Temperature and Oxygen Dependent Metabolite Utilization by Salmonella enterica Serovars Derby and Mbandaka

Salmonella enterica is a zoonotic pathogen of clinical and veterinary significance, with over 2500 serovars. In previous work we compared two serovars displaying host associations inferred from isolation statistics. Here, to validate genome sequence data and to expand on the role of environmental metabolite constitution in host range determination we use a phenotypic microarray approach to assess the ability of these serovars to metabolise ~500 substrates at 25°C with oxygen (aerobic conditions) to represent the ex vivo environment and at 37°C with and without oxygen (aerobic/anaerobic conditions) to represent the in vivo environment. A total of 26 substrates elicited a significant difference in the rate of metabolism of which only one, D-galactonic acid-g-lactone, could be explained by the presence (S. Mbandaka) or the absence (S. Derby) of metabolic genes. We find that S. Mbandaka respires more efficiently at ambient temperatures and under aerobic conditions on 18 substrates including: glucosominic acid, saccharic acid, trehalose, fumaric acid, maltotriose, N-acetyl-D-glucosamine, N-acetyl-beta-D-mannosamine, fucose, L-serine and dihydroxy-acetone; whereas S. Derby is more metabolically competent anaerobically at 37°C for dipeptides, glutamine-glutamine, alanine-lysine, asparagine-glutamine and nitrogen sources glycine and nitrite. We conclude that the specific phenotype cannot be reliably predicted from the presence of metabolic genes directly relating to the metabolic pathways under study.     

Molecular Characterization of Melanoma Cases in Denmark Suspected of Genetic Predisposition

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure. We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations. Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls. The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls. To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM. In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.     

DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves

The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272–422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.     

Soil-foraging animals alter the composition and co-occurrence of microbial communities in a desert shrubland

Animals that modify their physical environment by foraging in the soil can have dramatic effects on ecosystem functions and processes. We compared bacterial and fungal communities in the foraging pits created by bilbies and burrowing bettongs with undisturbed surface soils dominated by biocrusts. Bacterial communities were characterized by Actinobacteria and Alphaproteobacteria, and fungal communities by Lecanoromycetes and Archaeosporomycetes. The composition of bacterial or fungal communities was not observed to vary between loamy or sandy soils. There were no differences in richness of either bacterial or fungal operational taxonomic units (OTUs) in the soil of young or old foraging pits, or undisturbed soils. Although the bacterial assemblage did not vary among the three microsites, the composition of fungi in undisturbed soils was significantly different from that in old or young foraging pits. Network analysis indicated that a greater number of correlations between bacterial OTUs occurred in undisturbed soils and old pits, whereas a greater number of correlations between fungal OTUs occurred in undisturbed soils. Our study suggests that digging by soil-disturbing animals is likely to create successional shifts in soil microbial and fungal communities, leading to functional shifts associated with the decomposition of organic matter and the fixation of nitrogen. Given the primacy of organic matter decomposition in arid and semi-arid environments, the loss of native soil-foraging animals is likely to impair the ability of these systems to maintain key ecosystem processes such as the mineralization of nitrogen and the breakdown of organic matter, and to recover from disturbance.     

MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.