CDKN2A/p16 is inactivated in most melanoma cell lines

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Deletion mapping suggests that the 1p22 melanoma susceptibility gene is a tumor suppressor localized to a 9-Mb interval

Loss of the short arm of chromosome 1 is frequently observed in many tumor types, including melanoma. We recently localized a third melanoma susceptibility locus to chromosome band 1p22. Critical recombinants in linked families localized the gene to a 15-Mb region between D1S430 and D1S2664. To map the locus more finely we have performed studies to assess allelic loss across the region in a panel of melanomas from 1p22-linked families, sporadic melanomas, and melanoma cell lines. Eighty percent of familial melanomas exhibited loss of heterozygosity (LOH) within the region, with a smallest region of overlapping deletions (SRO) of 9 Mb between D1S207 and D1S435. This high frequency of LOH makes it very likely that the susceptibility locus is a tumor suppressor. In sporadic tumors, four SROs were defined. SRO1 and SRO2 map within the critical recombinant and familial tumor region, indicating that one or the other is likely to harbor the susceptibility gene. However, SRO3 may also be significant because it overlaps with the markers with the highest 2-point LOD score (D1S2776), part of the linkage recombinant region, and the critical region defined in mesothelioma. The candidate genes PRKCL2 and GTF2B, within SRO2, and TGFBR3, CDC7, and EVI5, in a broad region encompassing SRO3, were screened in 1p22-linked melanoma kindreds, but no coding mutations were detected. Allelic loss in melanoma cell lines was significantly less frequent than in fresh tumors, indicating that this gene may not be involved late in progression, such as in overriding cellular senescence, necessary for the propagation of melanoma cells in culture.

Precipitazioni sull'endotelio [Pigment Dispersion Syndrome]

Arguably, the most common patient seen in contact lens practice in our communities is the young adult white myope. The incidence of eye disease in this group of patients is very low, particularly if the genetically determined problems are excluded. However, there is one condition that should always be anticipated and searched for, especially in males. In fact, it affects 2-4% of these individuals and is known as pigment dispersion syndrome. This is important because 25-50% of these patients will get secondary or pigmentary glaucoma because of the pigment dispersion. It can be inherited as an autosomal dominant trait and has been mapped to chromosome 7. It is usually bilateral and is rarely encountered in patients of darker skin such as Asians and African-Americans.

Mitochondrial genome deletions and minicircles are common in lice (Insecta: Phthiraptera)

Background The gene composition, gene order and structure of the mitochondrial genome are remarkably stable across bilaterian animals. Lice (Insecta: Phthiraptera) are a major exception to this genomic stability in that the canonical single chromosome with 37 genes found in almost all other bilaterians has been lost in multiple lineages in favour of multiple, minicircular chromosomes with less than 37 genes on each chromosome. Results Minicircular mt genomes are found in six of the ten louse species examined to date and three types of minicircles were identified: heteroplasmic minicircles which coexist with full sized mt genomes (type 1); multigene chromosomes with short, simple control regions, we infer that the genome consists of several such chromosomes (type 2); and multiple, single to three gene chromosomes with large, complex control regions (type 3). Mapping minicircle types onto a phylogenetic tree of lice fails to show a pattern of their occurrence consistent with an evolutionary series of minicircle types. Analysis of the nuclear-encoded, mitochondrially-targetted genes inferred from the body louse, Pediculus, suggests that the loss of mitochondrial single-stranded binding protein (mtSSB) may be responsible for the presence of minicircles in at least species with the most derived type 3 minicircles (Pediculus, Damalinia). Conclusions Minicircular mt genomes are common in lice and appear to have arisen multiple times within the group. Life history adaptive explanations which attribute minicircular mt genomes in lice to the adoption of blood-feeding in the Anoplura are not supported by this expanded data set as minicircles are found in multiple non-blood feeding louse groups but are not found in the blood-feeding genus Heterodoxus. In contrast, a mechanist explanation based on the loss of mtSSB suggests that minicircles may be selectively favoured due to the incapacity of the mt replisome to synthesize long replicative products without mtSSB and thus the loss of this gene lead to the formation of minicircles in lice.

Physical and linkage maps for Drosophila serrata, a model species for studies of clinal adaptation and sexual selection

Drosophila serrata is a member of the montium group, which contains more than 98 species and until recently was considered a subgroup within the melanogaster group. This Drosophila species is an emerging model system for evolutionary quantitative genetics and has been used in studies of species borders, clinal variation and sexual selection. Despite the importance of D. serrata as a model for evolutionary research, our poor understanding of its genome remains a significant limitation. Here, we provide a first-generation gene-based linkage map and a physical map for this species. Consistent with previous studies of other drosophilids we observed strong conservation of genes within chromosome arms homologous with D. melanogaster but major differences in within-arm synteny. These resources will be a useful complement to ongoing genome sequencing efforts and QTL mapping studies in this species

Human papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa : a cross-sectional study

Background Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. Methods A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. Results The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 – 57.7) for those that were HC2 positive and had a viral load of ≤ 181.1 RLU (the median HPV viral load), and 33.8 (95% CI 6.4 – 178.9) for those that were HC2 positive with a HPV viral load > 181.1 RLU. Conclusion Women initiating ARVs have a high prevalence of abnormal Pap smears and HR-HPV. Our results underscore the need for locally relevant, rigorous screening protocols for the increasing numbers of women accessing ARV therapy so that the benefits of ARVs are not partially offset by an excess risk in cervical cancer.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

A meta-analysis of genome-wide association studies to identify prostate cancer susceptibility loci associated with aggressive and non-aggressive disease

Genome-wide association studies (GWAS) have identified multiple common genetic variants associated with an increased risk of prostate cancer (PrCa), but these explain less than one-third of the heritability. To identify further susceptibility alleles, we conducted a meta-analysis of four GWAS including 5953 cases of aggressive PrCa and 11 463 controls (men without PrCa). We computed association tests for approximately 2.6 million SNPs and followed up the most significant SNPs by genotyping 49 121 samples in 29 studies through the international PRACTICAL and BPC3 consortia. We not only confirmed the association of a PrCa susceptibility locus, rs11672691 on chromosome 19, but also showed an association with aggressive PrCa [odds ratio = 1.12 (95% confidence interval 1.03-1.21), P = 1.4 × 10(-8)]. This report describes a genetic variant which is associated with aggressive PrCa, which is a type of PrCa associated with a poorer prognosis.

The human tissue kallikrein and kallikrein-related peptidase family

The human kallikrein-related peptidases are a subgroup of trypsin and chymotrypsin-like serine peptidases that are characterized by their homology to tissue kallikrein or kallikrein 1 (KLK1) encoded by the KLK1 gene (reviewed in[1-4]). The human KLK locus spans an approximately 320 kb region on chromosome 19q13.3-13.4 and contains fifteen genes encoding KLK1 and fourteen other kallikrein-related peptidases, KLK2-KLK15, which have been named contiguously in the locus in the order of their discovery [5-8] (Figure 606.1). It is the largest contiguous cluster of serine protease encoding genes in the human genome which has evolved from gene duplication of KLK1 and then subsequent reduplication of the newly evolved KLK genes [2]. The high conservation noted for KLK1-KLK3 (62-77%) reflects the proposed duplication of the KLK1 gene that produced the KLK2 gene which further generated the KLK3 gene. In contrast, the newer KLK4-KLK15 proteases share much less similarity, from 24-66%, although strong homology between KLK4 and KLK5, KLK9 and KLK11, and KLK10 and KLK12 suggests these genes are duplications of each other [2]...

Mapping quantitative trait loci in a wild population using linkage and linkage disequilibrium analyses

Historical information can be used, in addition to pedigree, traits and genotypes, to map quantitative trait locus (QTL) in general populations via maximum likelihood estimation of variance components. This analysis is known as linkage disequilibrium (LD) and linkage mapping, because it exploits both linkage in families and LD at the population level. The search for QTL in the wild population of Soay sheep on St. Kilda is a proof of principle. We analysed the data from a previous study and confirmed some of the QTLs reported. The most striking result was the confirmation of a QTL affecting birth weight that had been reported using association tests but not when using linkage-based analyses. Copyright © Cambridge University Press 2010.

Prediction of IBD based on population history for fine gene mapping

A novel multiple regression method (RM) is developed to predict identity-by-descent probabilities at a locus L (IBDL), among individuals without pedigree, given information on surrounding markers and population history. These IBDL probabilities are a function of the increase in linkage disequilibrium (LD) generated by drift in a homogeneous population over generations. Three parameters are sufficient to describe population history: effective population size (Ne), number of generations since foundation (T), and marker allele frequencies among founders (p). IBD L are used in a simulation study to map a quantitative trait locus (QTL) via variance component estimation. RM is compared to a coalescent method (CM) in terms of power and robustness of QTL detection. Differences between RM and CM are small but significant. For example, RM is more powerful than CM in dioecious populations, but not in monoecious populations. Moreover, RM is more robust than CM when marker phases are unknown or when there is complete LD among founders or Ne is wrong, and less robust when p is wrong. CM utilises all marker haplotype information, whereas RM utilises information contained in each individual marker and all possible marker pairs but not in higher order interactions. RM consists of a family of models encompassing four different population structures, and two ways of using marker information, which contrasts with the single model that must cater for all possible evolutionary scenarios in CM.

Mapping of multiple quantitative trait loci affecting bovine spongiform encephalopathy

A whole-genome scan was conducted to map quantitative trait loci (QTL) for BSE resistance or susceptibility. Cows from four half-sib families were included and 173 microsatellite markers were used to construct a 2835-cM (Kosambi) linkage map covering 29 autosomes and the pseudoautosomal region of the sex chromosome. Interval mapping by linear regression was applied and extended to a multiple-QTL analysis approach that used identified QTL on other chromosomes as cofactors to increase mapping power. In the multiple-QTL analysis, two genome-wide significant QTL (BTA17 and X/Y ps) and four genome-wide suggestive QTL (BTA1, 6, 13, and 19) were revealed. The QTL identified here using linkage analysis do not overlap with regions previously identified using TDT analysis. One factor that may explain the disparity between the results is that a more extensive data set was used in the present study. Furthermore, methodological differences between TDT and linkage analyses may affect the power of these approaches.

Genome-wide search for markers associated with bovine spongiform encephalopathy

A genome-wide search for markers associated with BSE incidence was performed by using Transmission-Disequilibrium Tests (TDTs). Significant segregation distortion, i.e., unequal transmission probabilities of alleles within a locus, was found for three marker loci on Chromosomes (Chrs) 5, 10, and 20. Although TDTs are robust to false associations owing to hidden population substructures, it cannot distinguish segregation distortion caused by a true association between a marker and bovine spongiform encephalopathy (BSE) from a population-wide distortion. An interaction test and a segregation distortion analysis in half-sib controls were used to disentangle these two alternative hypotheses. None of the markers showed any significant interaction between allele transmission rates and disease status, and only the marker on Chr 10 showed a significant segregation distortion in control individuals. Nevertheless, the control group may have been a mixture of resistant and susceptible but unchallenged individuals. When new genotypes were generated in the vicinity of these three markers, evidence for an association with BSE was confirmed for the locus on Chr 5.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.