A Genome Wide Meta-Analysis Study for Identification of Common Variation Associated with Breast Cancer Prognosis

Objective

Genome wide association studies (GWAs) of breast cancer mortality have identified few potential associations. The concordance between these studies is unclear. In this study, we used a meta-analysis of two prognostic GWAs and a replication cohort to identify the strongest associations and to evaluate the loci suggested in previous studies. We attempt to identify those SNPs which could impact overall survival irrespective of the age of onset.

Methods

To facilitate the meta-analysis and to refine the association signals, SNPs were imputed using data from the 1000 genomes project. Cox-proportional hazard models were used to estimate hazard ratios (HR) in 536 patients from the POSH cohort (Prospective study of Outcomes in Sporadic versus Hereditary breast cancer) and 805 patients from the HEBCS cohort (Helsinki Breast Cancer Study). These hazard ratios were combined using a Mantel-Haenszel fixed effects meta-analysis and a p-value threshold of 5×10⁻⁸ was used to determine significance. Replication was performed in 1523 additional patients from the POSH study.

Results

Although no SNPs achieved genome wide significance, three SNPs have significant association in the replication cohort and combined p-values less than 5.6×10⁻⁶. These SNPs are; rs421379 which is 556 kb upstream of ARRDC3 (HR = 1.49, 95% confidence interval (CI) = 1.27–1.75, P = 1.1×10⁻⁶), rs12358475 which is between ECHDC3 and PROSER2 (HR = 0.75, CI = 0.67–0.85, P = 1.8×10⁻⁶), and rs1728400 which is between LINC00917 and FOXF1.

Conclusions

In a genome wide meta-analysis of two independent cohorts from UK and Finland, we identified potential associations at three distinct loci. Phenotypic heterogeneity and relatively small sample sizes may explain the lack of genome wide significant findings. However, the replication at three SNPs in the validation cohort shows promise for future studies in larger cohorts. We did not find strong evidence for concordance between the few associations highlighted by previous GWAs of breast cancer survival and this study.     

Mutation Pattern Variation Among Regions of the Primate Genome

We sequenced three argininosuccinate-synthetase-processed pseudogenes (ΨAS-A1, ΨAS-A3, ΨAS-3) and their noncoding flanking sequences in human, orangutan, baboon, and colobus. Our data showed that these pseudogenes were incorporated into the genome of the Old World monkeys after the divergence of the Old World and New World monkey lineages. These pseudogene flanking regions show variable mutation rates and patterns. The variation in the G/C to A/T mutation rate (u) can account for the unequal GC contents at equilibrium: 34.9, 36.9, and 41.7% in the pseudogene ΨAS-A1, ΨAS-A3, and ΨAS-3 flanking regions, respectively. The A/T to G/C mutation rate (v) seems stable and the u/v ratios equal 1.9, 1.7, and 1.4 in the flanking regions of ΨAS-A1, ΨAS-A3, and ΨAS-3, respectively. These ``regional' variations of the mutation rate affect the evolution of the pseudogenes, too. The ratio u/v being greater than 1.0 in each case, the overall mutation rate in the GC-rich pseudogenes is, as expected, higher than in their GC-poor flanking regions. Moreover, a ``sequence effect' has been found. In the three cases examined u and v are higher (at least 20%) in the pseudogene than in its flanking region—i.e., the pseudogene appears as mutation ``hot' spots embedded in ``cold' regions. This observation could be partly linked to the fact that the pseudogene flanking regions are long-standing unconstrained DNA sequences, whereas the pseudogenes were relieved of selection on their coding functions only around 30–40 million years ago. We suspect that relatively more mutable sites maintained unchanged during the evolution of the argininosuccinate gene are able to change in the pseudogenes, such sites being eliminated or rare in the flanking regions which have been void of strong selective constraints over a much longer period. Our results shed light on (1) the multiplicity of factors that tune the spontaneous mutation rate and (2) the impact of the genomic position of a sequence on its evolution. Received: 10 February 1997 / Accepted: 21 April 1997     

Mapping the Constrained Coding Regions in the Human Genome to Their Corresponding Proteins

Constrained Coding Regions (CCRs) in the human genome have been derived from DNA sequencing data of large cohorts of healthy control populations, available in the Genome Aggregation Database (gnomAD) [1]. They identify regions depleted of protein-changing variants and thus identify segments of the genome that have been constrained during human evolution. By mapping these DNA-defined regions from genomic coordinates onto the corresponding protein positions and combining this information with protein annotations, we have explored the distribution of CCRs and compared their co-occurrence with different protein functional features, previously annotated at the amino acid level in public databases.As expected, our results reveal that functional amino acids involved in interactions with DNA/RNA, protein–protein contacts and catalytic sites are the protein features most likely to be highly constrained for variation in the control population. More surprisingly, we also found that linear motifs, linear interacting peptides (LIPs), disorder–order transitions upon binding with other protein partners and liquid–liquid phase separating (LLPS) regions are also strongly associated with high constraint for variability. We also compared intra-species constraints in the human CCRs with inter-species conservation and functional residues to explore how such CCRs may contribute to the analysis of protein variants. As has been previously observed, CCRs are only weakly correlated with conservation, suggesting that intraspecies constraints complement interspecies conservation and can provide more information to interpret variant effects.     

水稻种子活力QTL定位及上位性分析

利用1个粳／籼交来源(Lemont／Teqing)、包含264个重组自交系的作图群体,采用纸卷法在18℃培养箱中进行2次重复的发芽实验,考察了种子发芽7d、9d和1ld的发芽率,种子发芽15d后的芽长及干重等种子活力的相关性状。结合一张含有198个DNA标记的连锁图谱,用作图软件QTLMapper1．0定位与种子活力相关的QTL。共检测到13个主效应QTL,这些QTL对性状的贡献率为2．9％～12．7％,平均贡献率为6．2％。同时检测到18对贡献率≥5％的互作位点,其贡献率为5．1％～11．8％,平均贡献率为6．9％,比检测到的主效应QTL的平均贡献率稍大。种子活力相关性状的大多数主效应和互作QTL成串分布于少数几个染色体区段(Chromosome Regions,CRs),并且成串分布在同一染色体区段的QTL效应的方向总是一致,该结果与这些性状在表型上的正相关相一致。若将成串分布有3个及3个以上种子活力相关性状QTL的CRs视为与种子活力高度相关的CRs,则共检测到7个上述与种子活力高度相关的CRs,分别分布在水稻12条染色体中的7条染色体上。根据所含QTL的种类(主效应QTL或／和上位性QTL)可将这些CRs分成以下3种：1)M-CRs：只含有主效应QTL,如CR^sv-7;2)E-CRs：所含位点没有主效应,但与其他位点发生互作,如CR^sv-1、CR^sv-6和CR^sv-12;3)ME-CRs：既含有主效应QTL、也含有与其他位点产生互作的互作位点,如CR^sv-2、CR^sv-5和CR^sv-8。另外还发现,有的CR上的位点同时与多个不同CR上的位点互作,影响种子活力的相关性状。与前入的研究结果相比较,发现有些与种子活力高度相关的CR可在不同研究者所用的不同定位群体中被检测到,而有的CR只在特定的定位群体中被检测到。由此表明,水稻种子活力具有丰富的遗传多样性和复杂的遗传基础,其主效QTL和互作位点可能基于遗传背景的不同而相互转化。     

Bacterial Populations Associated with Different Regions of the Human Colon Wall

The microorganisms associated with the undiseased human colon wall were examined in material obtained from four sudden-death victims. In traffic accident subjects (aged 45 and 16 years) the anaerobe-aerobe ratio was about 10⁴:1 in all areas of the colon examined, whereas in acute heart failure subjects (aged 74 and 46 years) the ratio was as low as 1.2:1. The flora of each individual was distinct and complex. Although the predominant anaerobes isolated were Bacteroides and Fusobacterium spp., which composed over 50% of the flora in some samples, the species isolated (indicated by morphology and glucose fermentation products) varied between individuals. Other major types observed were gram-positive nonsporing rods, including Bifidobacterium spp., and anaerobic cocci (between 8 and 20% of isolates). Clostridia were only isolated in significant numbers from one individual. Scanning electron microscopy showed that most of the organisms were present below the top surface of the mucin layer overlying the mucosa. The use of several different preparative procedures for microscopy showed a complex microbial structure within the mucus, but major variations in the bacterial populations in different areas of the colon were not found. Spiral-shaped organisms up to 60 μm long in the form of double helices were found in two subjects by scanning electron microscopy but were not isolated during the parallel bacteriological investigation. The differences between this and previous studies are discussed in relation to experimental procedures and also in contrast to results with animals that showed a particularly specialized flora associated with the colon wall.     

Evolution of MIR Elements Located in the Coding Regions of Human Genome

A search for new members of the mammalian interspersed repeat (MIR) family has been done over the coding regions of human genome from GenBank-116. Only 254 nucleotide sequences contained MIRs in coding regions, of which 45 MIR copies were unknown before, including 17 that occurred in translated gene regions. The program developed by the authors has been demonstrated to surpass the CENSOR program in the search power. The evolution of the MIR copies located in translated regions of human genome is discussed.     

Identification of Escherichia coli O157:H7 Genomic Regions Conserved in Strains with a Genotype Associated with Human Infection

Beta-glucuronidase-negative, sorbitol-nonfermenting isolates of Shiga toxin-producing Escherichia coli O157 comprise part of a clone complex of related enterohemorrhagic E. coli isolates. High-resolution genotyping shows that the O157 populations have diverged into two different lineages that appear to have different ecologies. To identify genomic regions unique to the most common human-associated genotype, suppression subtractive hybridization was used to identify DNA sequences present in two clinical strains representing the human lineage I O157:H7 strains but absent from two bovine-derived lineage II strains. PCR assays were then used to test for the presence of these regions in 10 lineage I strains and 20 lineage II strains. Twelve conserved regions of genomic difference for lineage I (CRD_I) were identified that were each present in at least seven of the lineage I strains but absent in most of the lineage II strains tested. The boundaries of the lineage I conserved regions were further delimited by PCR. Eleven of these CRD_I were associated with E. coli Sakai S-loops 14, 16, 69, 72, 78, 82, 83, 91 to 93, 153, and 286, and the final CRD_I was located on the pO157 virulence plasmid. Several potential virulence factors were identified within these regions, including a putative hemolysin-activating protein, an iron transport system, and several possible regulatory genes. Cluster analysis based on lineage I conserved regions showed that the presence/absence of these regions was congruent with the inferred phylogeny of the strains.     

Identification of Accessory Genome Regions in Poultry Clostridium perfringens Isolates Carrying the netB Plasmid

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.     

Identification of Fungal Species Associated with Cladode Spot of Prickly Pear and Their Sensitivity to Chitosan

México is the most important producer of prickly pear (Opuntia ficus‐indica) in the world. There are several fungal diseases that can have a negative impact on their yields. In this study, there was a widespread fungal richness on cladodes spot of prickly pears from México. A total of 41 fungi isolates were obtained from cladodes spot; 11 of them were morphologically different. According to the pathogenicity test, seven isolates caused lesions on cladodes. The morphological and molecular identification evidenced the isolation of Colletotrichum gloeosporioides, Alternaria alternata, Fusarium lunatum, Curvularia lunata. All these species caused similar symptoms of circular cladodes spot. However, it is noticeable that some lesions showed perforation and detachment of affected tissues by Fusarium lunatum. To our knowledge, this is the first report of the Fusarium lunatum as phytopathogenic fungus of cladodes of prickly pear. The chitosan inhibited the mycelium growth in the seven isolates of phytopathogenic fungi. Chitosan applications diminished the disease incidence caused by C. gloeosporioies and F. lunatum in 40 and 100%, respectively. Likewise, the lesion severity index in cladodes decreased. There are no previous reports about the application of chitosan on cladodes of prickly pears for the control of phytopathogenic fungi. Therefore, this research could contribute to improve the strategies for the management of diseases in prickly pear.     

Centromere-Like Regions in the Budding Yeast Genome

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres.     

Membrane Mutation Associated with Sensitivity to Acriflavine in Escherichia coli

The role of plasma membrane on the acriflavine sensitivity of Escherichia coli was studied. (14)C-uracil incorporation into ribonucleic acid fraction by spheroplasts was more sensitive to acriflavine in the acriflavine-sensitive strain (genotype acrA) than in the acriflavine-resistant (genotype acrA(+)) strain. There was no difference between two types of cells in the response to osmotic shock, phage sensitivity, and other treatments used to investigate the structure and stability of cell wall. Differences in the electron-microscopic figures between acrA and acrA(+) cells was found in the plasma membrane, surface area just below the membrane, and ribosomal aggregation, when cells were treated with acriflavine. It is concluded that a primary site of acriflavine action is on the plasma membrane, and the acrA mutation is mediated by it. On the basis of the present results, it is evident that differences in the acriflavine binding and the sensitivity to phenethyl alcohol and sodium dodecyl sulfate between the acrA and acrA(+) strains, previously reported, are attributable to a structural difference in the plasma membrane between the two strains.