The waddlia genome: A window into chlamydial biology

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2′116′312bp chromosome and a 15′593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.

Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species

Background Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species. Results We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes). Conclusions We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.

Common variation in the fibroblast growth factor receptor 2 gene is not associated with endometriosis risk

BACKGROUND Endometriosis is a polygenic disease with a complex and multifactorial aetiology that affects 8-10% of women of reproductive age. Epidemiological data support a link between endometriosis and cancers of the reproductive tract. Fibroblast growth factor receptor 2 (FGFR2) has recently been implicated in both endometrial and breast cancer. Our previous studies on endometriosis identified significant linkage to a novel susceptibility locus on chromosome 10q26 and the FGFR2 gene maps within this linkage region. We therefore hypothesized that variation in FGFR2 may contribute to the risk of endometriosis. METHODS We genotyped 13 single nucleotide polymorphisms (SNPs) densely covering a 27 kb region within intron 2 of FGFR2 including two SNPs (rs2981582 and rs1219648) significantly associated with breast cancer and a total 40 tagSNPs across 150 kb of the FGFR2 gene. SNPs were genotyped in 958 endometriosis cases and 959 unrelated controls. RESULTS We found no evidence for association between endometriosis and FGFR2 intron 2 SNPs or SNP haplotypes and no evidence for association between endometriosis and variation across the FGFR2 gene. CONCLUSIONS Common variation in the breast-cancer implicated intron 2 and other highly plausible causative candidate regions of FGFR2 do not appear to be a major contributor to endometriosis susceptibility in our large Australian sample.

Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines

The CDKN2 gene, encoding the cyclin dependent kinase inhibitor p16, is a tumour suppressor gene involved in melanoma and maps to chromosome band 9p22. Mutations or interstitial deletions of this gene have been found both in the germline of familial melanoma cases and somatically in melanoma cell lines. Previous mutation analyses of melanoma cell lines have indicated a high frequency of C:G to T:A transitions, with all of these mutations occurring at dipyrimidine sites. Including three melanoma cell lines carrying tandem CC to TT mutations, the spectrum of mutations so far reported indicates a possible role for u.v. radiation in the mutagenesis of this gene in some tumours. To further examine this hypothesis we have characterised mutations of the CDKN2 gene in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous deletions of the gene. Of the remaining cell lines, eight were shown by direct sequencing of PCR products from exon 1 and exon 2 to carry a total of nine different mutations of CDKN2. Two cell lines carried tandem CC to TT mutations and a high rate of C:G to T:A transitions was observed. This study provides further evidence for the role of u.v. light in the genesis of melanoma, with one target being the CDKN2 tumour suppressor gene.

Cloning and characterization of a novel human gene related to vascular endothelial growth factor

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

Compilation of somatic mutations of the CDKN2 gene in human cancers : non-random distribution of base substitutions

The CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, is a tumour suppressor gene that maps to chromosome band 9p21-p22. The most common mechanism of inactivation of this gene in human cancers is through homozygous deletion; however, in a smaller proportion of tumours and tumour cell lines intragenic mutations occur. In this study we have compiled a database of over 120 published point mutations in the CDKN2 gene from a wide variety of tumour types. A further 50 deletions, insertions, and splice mutations in CDKN2 have also been compiled. Furthermore, we have standardised the numbering of all mutations according to the full-length 156 amino acid form of p16. From this study we are able to define several hot spots, some of which occur at conserved residues within the ankyrin domains of p16. While many of the hotspots are shared by a number of cancers, the relative importance of each position varies, possibly reflecting the role of different carcinogens in the development of certain tumours. As reported previously, the mutational spectrum of CDKN2 in melanomas differs from that of internal malignancies and supports the involvement of UV in melanoma tumorigenesis. Notably, 52% of all substitutions in melanoma-derived samples occurred at just six nucleotide positions. Nonsense mutations comprise a comparatively high proportion of mutations present in the CDKN2 gene, and possible explanations for this are discussed.

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

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.