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Rare key functional domain missense substitutions in MRE11A, RAD50, and NBNcontribute to breast cancer susceptibility: results from a Breast Cancer Family Registry case-control mutation-screening study
- Francesca Damiola†1, 2,
- Maroulio Pertesi†1,
- Javier Oliver†1,
- Florence Le Calvez-Kelm1,
- Catherine Voegele1,
- Erin L Young3,
- Nivonirina Robinot1,
- Nathalie Forey1,
- Geoffroy Durand1,
- Maxime P Vallée1,
- Kayoko Tao3,
- Terrell C Roane4,
- Gareth J Williams5,
- John L Hopper6, 13,
- Melissa C Southey7,
- Irene L Andrulis8,
- Esther M John9, 12,
- David E Goldgar10,
- Fabienne Lesueur1, 11 and
- Sean V Tavtigian3Email author
DOI: 10.1186/bcr3669
© Damiola et al.; licensee BioMed Central Ltd. 2014
Received: 21 October 2013
Accepted: 8 May 2014
Published: 3 June 2014
The MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited mutations in the three components predispose to genetic instability disorders and the MRN genes have been implicated in breast cancer susceptibility, but the underlying data are not entirely convincing. Here, we address two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/BRCA2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions?
Using high-resolution melt curve analysis followed by Sanger sequencing, we mutation screened the coding exons and proximal splice junction regions of the MRN genes in 1,313 early-onset breast cancer cases and 1,123 population controls. Rare variants in the three genes were pooled using bioinformatics methods similar to those previously applied to ATM, BRCA1, BRCA2, and CHEK2, and then assessed by logistic regression.
Re-analysis of our ATM, BRCA1, and BRCA2 mutation screening data revealed that these genes do not harbor pathogenic alleles (other than modest-risk SNPs) with minor allele frequencies >0.1% in Caucasian Americans, African Americans, or East Asians. Limiting our MRN analyses to variants with allele frequencies of <0.1% and combining protein-truncating variants, likely spliceogenic variants, and key functional domain rare missense substitutions, we found significant evidence that the MRN genes are indeed intermediate-risk breast cancer susceptibility genes (odds ratio (OR) = 2.88, P = 0.0090). Key domain missense substitutions were more frequent than the truncating variants (24 versus 12 observations) and conferred a slightly higher OR (3.07 versus 2.61) with a lower P value (0.029 versus 0.14).
These data establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes. Like ATM and CHEK2, their spectrum of pathogenic variants includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study.
Based on risk and frequency, three classes of breast cancer susceptibility genes or loci are currently recognized: high-risk genes such as BRCA1 and BRCA2 (Mendelian Inheritance in Man numbers (MIMs) 113705 and 600185) in which protein-truncating mutations and severely dysfunctional missense substitutions confer a five- to ten-fold increased risk and for which the summed allele frequency in the general population is <1%; intermediate-risk genes such at ATM and CHEK2 (MIMs 208900 and 604373) in which protein-truncating mutations and severely dysfunctional missense substitutions confer a two- to five-fold increased risk and for which the summed allele frequency in the general population may approach 1%; and common, modest-risk single nucleotide polymorphisms (SNPs) which individually have much higher frequency but only rarely confer risk greater than 1.25-fold [1, 2]. Linkage analysis provided an effective genome-wide approach for locating high-risk susceptibility genes, and genome-wide association study provided an effective approach for finding risk-associated SNPs. While exome sequencing-based strategies may eventually provide a hypothesis-free, genome-wide approach for identification of intermediate-risk genes, much of our current knowledge base has flowed from candidate gene studies [3].
The MRN complex, formed from dimers of the proteins encoded by MRE11A, RAD50, and NBN (MIMs 600814, 604040, and 602667), plays key roles in DNA double-strand break (DSB) repair, meiotic recombination, cell cycle checkpoints, and maintenance of telomeres [4]. In mice, homozygous knockouts of these genes are lethal [5–7]. Humans born with biallelic mutations in any one of the three genes share a cellular phenotype that includes sensitivity to ionizing radiation, a deficit in DNA DSB repair, and chromosomal instability (MIM 604391; MIM 251260) [8]. Moreover, these people are at risk of severe cancer susceptibility phenotypes. For example, two brothers who were compound heterozygotes for mutations that fell in the amino region of the MRE11A protein died of pulmonary adenocarcinoma before age 20 [9, 10], which would seem very unlikely in the absence of an underlying cancer predisposition. Susceptibility to lymphoma is a prominent feature of Nijmegen breakage syndrome, which is caused by biallelic mutation of NBN[11]. While too few human biallelic RAD50 mutation carriers have been identified to reach a conclusion about their cancer susceptibility, more than 20% of mice homozygous for a hypomorphic Rad50 allele (Rad50 p.Lys22Met) that lived past age four months died with lymphoma or leukemia [12].
Breast cancer risks for heterozygous carriers of MRN gene mutations were summarized briefly by Hollestelle et al. [3]. Of the three genes, NBN has the strongest evidence in support of acting as an intermediate-risk breast cancer gene. This is largely because the truncating variant NBN c.657del5 has a high enough frequency among individuals of Slavic origin to be evaluated by case-control analysis, and meta-analysis of nine such studies revealed a combined odds ratio (OR) of 2.63 (95% confidence interval (CI) 1.76 to 3.93) for this variant [13]. Most evidence in favor of RAD50 rested on a truncating variant RAD50 c.687delT which has been observed in Finnish cases and controls [14, 15]; while subsequent studies in the same and other populations are consistent with the hypothesis that RAD50 is an intermediate-risk breast or pancreatic cancer susceptibility gene, they have not provided significant supporting evidence [16, 17]. Evidence for MRE11A rests primarily on the observation of two mutations in the gene from a series of eight non-BRCA1/2 breast cancer families with tumors that showed loss of all three MRN proteins [18].
Previously, we performed case-control mutation screening studies of ATM, CHEK2, XRCC2, and RAD51 to clarify our understanding of their role in breast cancer susceptibility [19–22]. A common thread across these studies has been use of bioinformatic and statistical approaches designed to detect evidence of pathogenicity from both truncating and splice junction variants (T + SJV) and/or rare missense substitutions (rMS). Here, we apply a case-control mutation screening strategy in an ethnically diverse series of subjects to evaluate MRE11A, RAD50, and NBN. Given that the three proteins form an evolutionarily conserved complex involved in maintenance of genomic integrity, we decided to evaluate the three genes as a single large candidate intermediate-risk breast cancer susceptibility gene with a concatenated open reading frame of 2,774 amino acids - which nevertheless is not quite as large as the 3,056 amino acid open reading frame of ATM. Our analysis addresses two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions?
Study sample
The design for this study has been described in detail previously [19, 20, 22]. Briefly, eligible participants included women ascertained by population-based sampling by the Australian, Northern California, and Ontario sites of the Breast Cancer Family Registry (BCFR) [23]. Subjects were recruited between 1995 and 2005. Selection criteria for cases (N = 1,313) were diagnosis of breast cancer at or before age 45 years and self-reported race/ethnicity plus grandparents' country of origin information consistent with Caucasian, East Asian, Hispanic/Latino, or African American racial/ethnic heritage. The controls (N = 1,123) were frequency matched to the cases within each center on racial/ethnic group, with age at selection not more than ± 10 years from the age range at diagnosis of the cases gathered from the same center. Because of the shortage of available controls in some racial/ethnic and age groups, the frequency matching was not one-to-one in all subgroups.
Recruitment and genetic studies were approved by the Ethics Committee of the International Agency for Research on Cancer (IARC), the University of Utah Institutional Review Board (IRB), and the local IRBs of the BCFR centers from which we received samples. These local IRBs were the Health Sciences Human Research Ethics Subcommittee of the University of Melbourne, Australia; the Institutional Review Board of the Cancer Prevention Institute of California; and the Research Ethics Boards of Mount Sinai Hospital and the University Health Network, Ontario, Canada. Written informed consent was obtained from each participant.
Mutation screening
For mutation screening of the coding exons and proximal splice junction regions of MRE11A (NM_005591.3), RAD50 (NM_005732.3) and NBN (NM_002485.4), we used 30 ng of whole-genome amplified (WGA) DNA obtained by mixing 15 ng of amplified DNA from each of two independent WGA reactions. The laboratory process was as described in detail for our recent studies of ATM, CHEK2, XRCC2, and RAD51[19–22]. Our semi-automated approach, handled by a Laboratory Information Management System (LIMS) [24, 25], relies on mutation scanning by high-resolution melt curve (HRM) analysis followed by direct Sanger sequencing of the individual samples for which an aberrant melting curve profile is indicative of the presence of a sequence variant. In a previous work, we showed that the HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening by comparing the results with those obtained with Sanger sequencing [26].
For MRN amplicons harboring a SNP(s) with frequency ≥1% in either dbSNP or initial amplicon testing, we applied a simultaneous mutation scanning and genotyping approach using HRM analysis to improve the sensitivity and the efficiency of the mutation screening, as described previously [24, 25].
All exonic sequence variants, plus intronic sequence variants that fell within 20 bp of a splice acceptor or eight bp of a splice donor, and were either unreported or had an allele frequency of <1% in the large scale reference groups 'Caucasian Americans', 'African Americans' and 'East Asians' based on exome variant server (EVS) and 1,000 genomes project (1000G) data [27, 28], were confirmed either by independent re-amplification and sequencing from each of the two independent WGA reaction products and concordant variant calls, or, for five variants, by re-amplification and sequencing from genomic DNA.
All samples that failed either at the primary PCR, secondary PCR, or sequencing reaction stage were re-amplified from WGA DNAs or genomic DNAs. Samples that still did not provide satisfactory mutation screening results for at least 80% of the concatenated MRN coding sequence were excluded from further analysis. Primer and probe sequences are available from the authors upon request.
Alignments and scoring of missense substitutions
We used M-Coffee [29], which is part of the Tree-based consistency objective function for alignment evaluation (T-Coffee) software suite of alignment tools [30] to prepare a protein multiple sequence alignment for each of the three MRN proteins (MRE11A, RAD50 and NBN) in order to predict the effect of missense substitutions on the proteins and on the activity of the MRN complex. Each alignment consisted of sequences from 16 species: Homo sapiens, Macaca mulatta, Callithrix jacchus, Mus musculus, either Sus scrofa or Bos Taurus, Loxodonta africana, Dasypus novemcinctus, Monodelphis domestica, Ornithorhynchus anatinus, Gallus gallus, Anolis carolinensis, either Xenopus tropicalis or Xenopus laevis, Latimeria chalumnae, either Fugu rubripes or Danio rerio, Branchiostoma lanceolatum, and Strongylocentrotus purpuratus. The alignments were characterized using the Protpars routine of Phylogeny Inference Package version 3.2 software (PHYLIP) [31] to make a maximum parsimony estimate of the number of substitutions that occurred along each clade of the underlying phylogeny. The sequence alignment, or updated versions thereof, is available at the Align Grantham variation Grantham deviation (Align-GVGD) website [32]. Missense substitutions observed during our mutation screening of the three MRN genes were scored using the Align-GVGD and Sorting Intolerant from Tolerant (SIFT) software programs with our curated alignments, and with polymorphism phenotyper (PolyPhen-2.1) software using its precompiled alignments [33–35]. In brief, Align-GVGD grades missense substitutions against a protein multiple sequence alignment based on a combination of Grantham Variation (GV), which measures the amount of physicochemical variation at a particular position in the alignment, and Grantham Deviation (GD), which measures the physicochemical difference between the missense residue and the range of variation observed at its position in the protein. The classifier provides seven ordered grades (C65, C55, C45, C35, C25, C15 and C0) ranging from the most likely deleterious to least likely deleterious [33]. SIFT is a sequence homology-based tool that predicts variants in the query sequence as 'tolerated' or 'deleterious' using normalized probabilities calculated from the input multiple sequence alignment [34]. Variants at a position with normalized probabilities less than 0.05 are predicted deleterious and predicted neutral with a probability greater than or equal to 0.05. PolyPhen-2 predicts variants as 'benign', 'possibly damaging', or 'probably damaging' based on eight sequence-based and three structure-based predictive features [35]. The alignment pipeline used in PolyPhen-2 selects homologous sequences using a clustering algorithm and then constructs and refines the alignment yielding an alignment containing both orthologs and paralogs that may or may not be full length, which yields a wider breadth of sequences but decreased depth compared with the curated alignments used with Align-GVGD and SIFT [36].
In silicoprediction on splicing
Sequence variants falling in the first three bp or last three bp of an exon, plus intronic variants detected in the vicinity of the splice junction sequences, with allele frequencies <1%, were scored for their potential impact on splicing using MaxEntScan (MES), which computes the maximum entropy score of a given sequence using splice site models trained on human data [37]. In work to be published elsewhere, we calibrated MES by calculating the average and standard deviation of MES scores for the wild-type splice junctions in BRCA1, BRCA2, and ATM, allowing us to convert raw MES scores into z-scores. Based on BRCA1 and BRCA2 mutation screening data used previously to calibrate Align-GVGD [33, 38], we found that rare variants that fall within the acceptor or donor region and reduce the MES score for the splice signal in which they fall show an approximately 95% probability to damage splice junction function when they result in a calibrated MES score of z < −2, or approximately 40% probability when they result in a calibrated MES score of −2 < z ≤ −1 [39]; Vallee et al., manuscript in preparation. Thus these MES-based rules were used to identify rare sequence variants that are likely to alter MRN gene mRNA splicing.
Analysis of rare variant threshold frequency from ATM, BRCA1, BRCA2, and CHEK2data
For ATM, we examined the relationship between sequence variant frequency and breast cancer risk as follows. Frequencies for all of the variants included in our 2009 ATM case-control mutation screening meta-analysis [19] were extracted from the EVS (for Caucasian American and African American frequencies) or UK National Genetics Reference Laboratory Manchester (NGRL Manchester) summary of 1000G data (for East Asian allele frequencies) [40]. T + SJV (as defined in [19]) plus missense substitutions and in-frame indels falling from position 1960 through the end of the protein were then grouped into the following allele frequency bins: 1% to 0.32%, 0.32% to 0.10%, 0.10% to 0.032%, <0.032%. ORs for each bin were then estimated by logistic regression, adjusting for study, ethnicity and mutation-screening method employed, using Stata version 11 software (StataCorp, College Station, TX, USA).
CHEK2 sequence variants included in our 2011 CHEK2 case-control mutation screening study [20] were evaluated in the same way except that (a) position in the protein was not considered, and (b) logistic regressions were adjusted for study center and race/ethnicity.
For BRCA1 and BRCA2, we examined the relationship between frequency and risk as follows. First we defined 'pseudo-cases' and 'pseudo-controls' from the tested population at Myriad Genetics in the data set we used to evaluate more than 1,000 BRCA1/2 sequence variants in 2007 [38]. The data set included results from approximately 68,000 full-sequence tests. Pseudo-cases for BRCA1 were defined to be all tested individuals who were affected with breast cancer and were not found to carry a pathogenic variant in either BRCA1 or BRCA2; pseudo-controls were taken to be affected individuals who were found to carry pathogenic mutations in BRCA2 (thus explaining their personal and family history of cancer). A similar approach was taken for BRCA2 variants, with carriers of BRCA1 pathogenic variants serving as pseudo-controls. For each gene, variants were categorized into frequency bins as described above. Logistic regression was then used to estimate ORs for each frequency bin. Note that, because carriage of clearly pathogenic variants was used to differentiate between pseudo-cases and pseudo-controls, the BRCA1 and BRCA2 ORs were estimated from the distributions of unclassified variants.
Statistical analyses of MRN case-control mutation screening data
To assess the relationship between MRN variants and breast cancer risk, analyses were performed using the chi-square test and multivariable unconditional logistic regression using Stata version 11 software (StataCorp). Differences in the case-control ratio between racial/ethnic groups and study center were accounted for by including categorical variables for each racial/ethnic group and each study center. Adjustment for racial/ethnic group should also capture confounding of genetic and social factors with interaction terms, allowing that this confounding effect may be different for the broadly labeled racial/ethnic groups in different centers. P values reported from analyses of the MRN case-control data are from the likelihood ratio test, adjusted for racial/ethnic group and study center, unless otherwise noted.
Number of subjects included in the analysis
Distribution of cases and controls by age, race or ethnicity, and study center †
Cases, n (%)
Controls, n (%)
Age range, yr
108 (8.2%)
67 (6.0%)
325 (24.8%)
172 (15.3%)
436 (33.2%)
237 (21.1%)
443 (33.8%)
203 (18.1%)
0 (0.0%)
230 (20.5%)
0 (0.0%)
212 (18.9%)
1,312 (100.0%)
1,121 (100.0%)
Race or ethnicity
848 (64.6%)
967 (86.3%)
East Asian
208 (15.9%)
71 (6.3%)
158 (12.0%)
47 (4.2%)
Recent African Ancestry
98 (7.5%)
36 (3.2%)
1,312 (100.0%)
1,121 (100.0%)
Study center
593 (45.2%)
522 (46.6%)
302 (23.0%)
463 (41.3%)
BCFR-Northern California
417 (31.8%)
136 (12.1%)
1,312 (100.0%)
1,121 (100.0%)
Relationship between frequency and odds ratio for homologous recombination repair pathway breast cancer susceptibility genes
Frequency vs odds ratio for confirmed HRR breast cancer susceptibility genes
Frequency bins (%) 1.000 - 0.320 OR (95% CI)
0.57 (0.12-2.80)
1.13 (0.30-4.24)
2.61 (1.57-4.35)
no variants
no variants
no variants
2.06 (1.06-4.57)
no variants
0.96 (0.64-1.43)
1.60 (0.95-2.70)
1.57 (1.15-2.15)
0.99 (0.20-4.91)
3.40 (1.22-9.47)
1.68 (0.29-9.78)
3.31 (1.49-7.37)
In the 0.32% to 0.10% allele frequency bin, there was significant evidence of pathogenic variants in CHEK2; indeed, the known pathogenic CHEK2 variants c.1100delC (Chr22:29091856delG) and p.Ile157Thr (rs17879961) both have allele frequencies between 0.32% and 0.10% in the EVS 'Caucasian American' sample. But there was no evidence for pathogenic variants in the other three genes in this allele frequency range.
In the 0.10 to 0.032% bin, BRCA2 had an OR of 1.60 (P = 0.078). Although just shy of significant, an elevated BRCA2 OR in this EVS frequency bin is supported by the presence of known pathogenic BRCA2 variants with multiple reports in the Breast Information Core (BIC) database such as c.2806_2809delAAAC (rs80359351) and c.3847_3848delGT (rs80359405) [41]. While the OR for ATM in this frequency bin was very near 1.0, our data set did contain one known pathogenic in-frame deletion in the gene, p.RIS2547_2549del (c.7638_7646del9), which was observed in a control.
Below an allele frequency of 0.032%, all four genes had significant evidence of pathogenic variants. We note that, in genotype-phenotype terms, the MRN genes more closely resemble BRCA1, BRCA2, and ATM than CHEK2 in that inheritance of biallelic mutations in the MRN genes is either embryonic lethal or causes a developmental phenotype that severely reduces reproductive fitness; we know of no such evidence for biallelic CHEK2 mutation carriers [42–44]. Therefore, noting that there was no evidence for pathogenic variants in ATM, BRCA1, or BRCA2 with allele frequencies >0.1% in continental level populations, we set our threshold for evaluating rare MRN variants at an allele frequency ≤0.1% in Caucasian Americans, African Americans, and East Asians, based on EVS data for the former two groups and 1000G data, as summarized by the NGRL Manchester, for East Asian allele frequencies [40].
Analyses of rare silent substitutions and rare, analytically innocuous splice junction variants
Analyses of largely innocuous groups of rare variants
Cases, n
Controls, n
Crude OR (95% CI)
Adjusted OR†(95% CI)
0.85 (0.35-2.06)
0.95 (0.38-2.39)
0.92 (0.43-1.96)
0.62 (0.28-1.39)
Any rMS or in-frame indel¥
1.08 (0.70-1.67)
0.96 (0.61-1.50)
Analysis of protein-truncating variants
Severity scores applied to selected classes of potentially pathogenic rare variants
Cases, n
Controls, n
Truncating and spliceogenic variants
Frameshift, excluding the last exon
Nonsense, excluding the last exon
Severe acceptor damage
Moderate acceptor damage
Severe donor damage
Moderate donor damage
Key domain rMS, graded C0
Key domain rMS, graded C15
Key domain rMS, graded C25
Key domain rMS, graded C35
Key domain rMS, graded C45
Key domain rMS, graded C55
Key domain rMS, graded C65
Key domain in-frame deletion†
Analyses of potentially pathogenic groups of rare variants
Cases, n
Controls, n
Crude OR†(95% CI)
Adjusted OR†(95% CI)
2.60 (0.70-9.65)
2.61 (0.67-10.1)
Any key functional domain rMS or in-frame indel*
4.18 (1.59-11.0)
3.17 (1.17-8.59)
Key functional domain rMS (severity > C0) or in-frame indel*
4.34 (1.48-12.7)
3.07 (1.01-9.31)
T + SJV¥ plus key functional domain rMS (severity > C0) or in-frame indel*
3.60 (1.57-8.24)
2.88 (1.22-6.78)
Analyses of rare missense substitutions and in-frame indels
We observed 58 distinct rMS, one in-frame deletion, and one final exon frameshift (RAD50 c.3852del4) that we treated as an in-frame deletion (Table S1 in Additional file 1). Taking into account that two subjects (one case and one control) carried two rMS, these added up to 48 cases and 38 controls that carried one or more rMS or in-frame indel. The OR for this class of variants was 0.96 (P = 0.85) (Table 3).
In our analyses of BRCA1 and BRCA2, we found that evidence for pathogenic, non-spliceogenic, missense substitutions in those genes was limited to substitutions that fall in the protein's key functional domains and have severity scores of greater than C0 when evaluated by Align-GVGD [33, 38]; that observation is also supported by an extensive functional assay analysis of BRCA1 missense substitutions [45]. In our case-control mutation screening analysis of ATM, we also found that evidence for rMS that predispose to breast cancer was limited to rMS with Align-GVGD severity scores of greater than C0 that fall in the key functional domains of the protein that are central to its enzymatic activity [19]. Accordingly, we carried out an analysis of MRN gene rMS limited to these key functional domains, largely as described in a recent review of MRN protein structure and function [4] with the exception that we excluded from the analysis variants falling in the second DNA binding domain of MRE11A because this domain is required for DSB formation during meiosis but not for repair of DSBs [46].
To refine analyses of the key domain rMS, we prepared and hand-curated protein multiple sequence alignments of the MRN proteins. Each alignment contains 16 sequences from MRN orthologs and, from the perspective of the position of Homo sapiens in the vertebrate phylogeny, each samples the same key nodes of mammalian and vertebrate evolution. The concatenated alignment slightly exceeds an average of 3.0 substitutions per position, reaching our criterion of sufficient sequence diversity for evaluating missense substitutions [36]. Using these alignments, the key domain rMS were scored with Align-GVGD and SIFT; PolyPhen-2 scores for these variants were also extracted from PolyPhen's pre-computed exome-wide data set [32, 64, 65]. The key domain final exon truncating variant RAD50 c.3852del4 was assigned Align-GVGD, SIFT, and PolyPhen-2 scores corresponding to the most severe missense substitution created by the frameshifted coding; these were C65, 0.00, and 0.996, respectively (Align-GVGD and PolyPhen-2 scales have the opposite polarity of the SIFT scale; all three of these scores are indicative of an extremely damaging variant). Accordingly, for all further analyses described here, RAD50 c.3852del4 is included in the most severe grade of key domain rMS unless otherwise noted.
A conservative view of protein multiple sequence alignment-based evaluation of human missense substitutions is that each missense substitution that falls within or very close to the range of variation observed at its position in an appropriately informative alignment should be neutral or nearly so. With Align-GVGD, this would correspond to missense substitutions that score C0. Using the complete alignments of the MRN proteins, 11 of the key domain rMS scored C0; these were carried by 11 cases and just one control, potentially indicative of overly stringent alignment depth. Reducing the stringency of the Align-GVGD scoring by restricting the alignments to the mammals-only subset (comprising sequences from nine species), four of the key domain rMS scored C0, and these were carried by four cases and one control. Excluding these four substitutions as likely innocuous, the 20 remaining key domain rMS were carried by 20 cases and four controls (Table 5 and Table S1 in Additional file 1). The OR for the non-C0 key domain rMS and in-frame deletions was 3.07 (P = 0.029).
Graded tests
ln(OR) regression coefficient*(95% CI)
Modeled OR at most severe grade†(95% CI)
Observed OR at most severe grade¥(95% CI)
MRN Key Domain missense, graded$
0.30 (0.055-0.55)
6.16 (1.39-27.3) ¢
4.31 (0.94-19.7)¢
0.21 (−0.052-0.48)
3.60 (0.73-17.7)¢
1.80 (0.38-8.44)¢
MRN T + SJV, graded
0.14 (−0.088-0.37)
2.34 (0.59-9.34)¢
2.03 (0.52-7.86)¢
0.15 (−0.091-0.38)
2.41 (0.58-10.0)¢
2.15 (0.53-8.78)¢
MRN T + SJV + KeyD missense, graded$
0.23 (0.059-0.39)
3.90 (1.42-10.7) ¢
2.93 (1.08-7.97) ¢
0.18 (0.0011-0.35)
2.89 (1.01-8.30) ¢
1.97 (0.69-5.62)¢
MRN T + SJV + KeyD missense, graded$ (C65 rMS and in-frame indel removed)
0.31 (0.0094-0.62)
3.50 (1.04-11.8) ∞
2.88 (0.79-10.5) ∞
0.33 (0.028-0.64)
3.79 (1.12-12.9) ∞
3.43 (0.91-12.9) ∞
Truncating variants and rare missense substitutions assessed in a single model
Using a simple binary classification to combine T + SJV plus key functional domain rMS of grade > C0 into a single model, we observed 29 carriers among the cases against seven among the controls. This binary classification resulted in an OR of 2.88, P = 0.0090 (Table 5).
To add the T + SJV into the regression test for OR trend across the graded rMS, we assigned protein-truncating variants a severity grade equal to that of the highest grade rMS included in that regression, in this case 6.0. Potentially damaging splice junction variants were assigned this severity grade x their probability to damage a splice junction, that is, severely damaging splice junction variants were assigned a severity grade of 0.95 × 6 = 5.7, and moderately damaging splice junction variants a severity grade of 0.40 × 6 = 2.4. This test yielded a lognormal OR increase of 0.18/grade (P = 0.033), which corresponds to a modeled OR of 2.89 at the most severe grade (Table 6).
In addition to the lognormal OR coefficient and modeled OR at the most severe sequence variant grade, Table 6 also reports the OR point estimate at the most severe sequence variant grade. One unusual result was that, for the analysis or rMS, the logistic regression adjustment for race/ethnicity and study center decreased the modeled OR for key domain C65 rMS by 42% and decreased the OR point estimated for this class of rMS by 58% compared to the crude result. These ORs also decreased in the combined test of rMS plus T + SJV, but not as dramatically.
Tests of heterogeneity
Crude (Fisher's exact)
Heterogeneity across racial/ethnic groups by sequence variant class
Racial/ethnic group: T + SJV
Racial/ethnic group: any rMS
subset: non-key domain rMS*:
subset: key domain rMS*:
sub-subset: key domain rMS*, <C65
sub-subset: key domain rMS*, C65
Heterogeneity across genes, by likely pathogenic sequence variant class
MRN: key domain rMS*
MRN: rare T + SJV and key domain rMS*
Impact of race/ethnicity heterogeneity in distribution of rare key domain C65 missense substitutions
Cases, n
Controls, n
Crude OR†(95% CI)
Adjusted OR†(95% CI)
Key functional domain* C65 rMS or in-frame indel§
4.35 (0.95-19.9)
1.78 (0.38-8.35)
Key functional domain* rMS (C0 < severity < C65)$
4.34 (0.95-19.9)
4.55 (0.97-21.3)
T + SJV¥ plus key functional domain* rMS (C0 < severity < C65)$
3.30 (1.23-8.87)
3.39 (1.23-9.33)
T + SJV¥ plus key functional domain* rMS (severity > C0), analysis limited to CEU¶ subjects.
4.04 (1.33-12.3)
4.08 (1.32-12.5)
Individual contributions of MRE11A , RAD50 , and NBN to the ensemble model
Cases, n
Controls, n
Crude OR†(95% CI)
Adjusted OR†(95% CI)
rMS or IFD*¢
6.08 (0.75-49.5)
3.62 (0.42-31.5)
6.95 (0.87-55.6)
5.02 (0.59-42.8)
1.16 (0.26-5.18)
1.09 (0.23-5.24)
rMS or IFD*
4.34 (0.95-19.9)
3.21 (0.68-15.2)
2.43 (0.87-6.77)
1.98 (0.68-5.71)
rMS or IFD*
2.60 (0.27-25.1)
2.12 (0.20-22.6)
6.08 (0.75-49.5)
5.28 (0.62-45.2)
MRE11A, RAD50, and NBN ensemble model*
2.60 (0.70-9.65)
2.61 (0.67-10.1)
rMS or IFD*¢
4.34 (1.48-12.7)
3.07 (1.01-9.31)
3.60 (1.57-8.24)
2.88 (1.22-6.78)
Although MRN gene T + SJV were not by themselves a significant breast cancer risk factor, we note that our OR point estimate of 2.61 is both very close to the meta-analysis point estimate of 2.63 that Zhang et al. obtained for NBN c.657del5 [13], and close to the point estimate of 2.32 that we obtained in our meta-analysis of ATM T + SJV [19]. Thus, while we cannot exclude that our nonsignificant finding is actually indicative of little or no risk of breast cancer conferred by MRN gene protein-truncating variants, our data are more strongly in accord with the hypothesis that they confer an intermediate risk of magnitude similar to the risk conferred by truncating variants in ATM.
Overall, there was no association between rMS and risk of breast cancer. Nevertheless, tightening the focus to key functional domain rMS resulted in a significant association with an OR of approximately 3.0. In this sense, the MRN genes behave as the homologous recombination repair genes BRCA1, BRCA2, and ATM - genes in which rare missense substitutions that are pathogenic because of missense dysfunction per se are largely confined to key functional domains.
Combining MRN T + SJV and key functional domain rMS, we observed an OR of 2.88 with a P value of 0.0090. That P value meets the threshold of P <0.01 that Hollestelle et al. suggested for establishing intermediate-risk susceptibility genes that were already strong candidates based on their biochemical function [3]. Thus with a mutation screening and data analysis approach that considered MRE11A, RAD50, and Nibrin as a unique functional entity and focused the analysis of rMS to those that fall in the key functional domains of the MRN complex, we overcame the limitation of previous suggestive studies that were based on a small number of founder mutations [13, 15, 18], and confirmed the hypothesis that MRE11A, RAD50, and NBN are intermediate-risk susceptibility genes in a general sense. Moreover, because we did not observe any of the four sequence variants most responsible for the MRN genes' candidate gene status (MRE11A p.Arg202Gly, MRE11A p.Arg633Stop, RAD50 c.687delT, and NBN c.657del5) [13, 14, 18], this confirmation is independent of the hypothesis-generating data.
Five of the sequence variants observed in the MRN case-control mutation screening bear further discussion.
NBN p.Arg215Trp was of interest because association studies have found evidence that it confers modest risk of several cancers (for review, see [66]), and there is biochemical evidence, albeit somewhat conflicting, of altered function of this nibrin allele [67, 68]. We observed six cases and six controls with the p.Arg215Trp missense substitution, resulting in an OR of 0.96 (95% CI 0.30 to 3.06, P = 0.95). While these confidence intervals are too wide to exclude the possibility that NBN p.Arg215Trp is actually a modest-risk variant, we also point out that position Arg215 is quite variable in our protein multiple sequence alignment and that, according to EVS data, the variant has a frequency of 0.37% in Caucasian Americans - well above the frequency threshold we found for severely dysfunctional variants in homologous recombination repair genes in which biallelic mutations cause embryonic lethality or severe childhood disease.
Second, we observed one carrier, a Caucasian control ascertained at the age of 55, of the MRE11A in-frame deletion c.2109del9. The variant falls in the last exon of the gene, near the carboxy terminus of the second DNA binding domain (which is also the carboxy terminus of the protein). Because this domain is required for double-strand break formation during meiosis but not for repair of double-strand breaks [46], the domain was not included in the list of 'key functional domains' and the indel was not included in statistical analyses of key functional domain variants.
Third, we observed one carrier, an East Asian case diagnosed at the age of 35, of MRE11A p.Thr481Ile. This residue is a threonine in all but one species in our alignment, but is a methionine in the cephalochordate, Branchiostoma lanceolatum. The substitution falls within the protein's RAD50 interaction domain. While very few of the rare variants that we observed have been reported in human ataxia-telangiectasia-like disease or Nijmegen breakage syndrome patients, another substitution at this residue, p.Thr481Lys, was observed in an Italian ataxia-telangiectasia-like disease sib-pair [69].
Fourth, we observed one carrier, an East Asian control ascertained at age 50, of the RAD50 frameshift c.3852del4. Because the frameshift falls in the last exon of the gene where it would not be expected to cause nonsense-mediated decay of the mRNA, we evaluated it as an in-frame deletion rather than as a frameshift. As such, it scrambles well-conserved sequence near the carboxy terminus of the protein's carboxy-end ATPase domain and final MRE11A binding domain including positions such as Arg1288 and Lys1291 that are invariant in our protein multiple sequence alignment. The sequence scrambling creates nonconservative substitutions at invariant key functional domain positions, resulting in the highest possible sequence variant severity score.
Fifth, we observed one carrier, also an East Asian control ascertained at age 50, of the NBN missense substitution p.Ile35Thr. This position falls in the protein's functionally important forkhead-associated (FHA) domain and is either isoleucine or leucine in all of the species included in our NBN protein multiple sequence alignment.
The last three variants described above illustrate two of the analytic problems encountered in this study. All three were evaluated as key domain C65 rMS and all three were observed in East Asian subjects. Combined with eight additional observations of key domain C65 rMS in either East Asian or Latina subjects against just one in a Caucasian of European ancestry, there was an unexpected excess of these variants in the non-Caucasian subjects mutation screened in this study. Second, the two variants observed in the controls affected positions with little or no cross-species physicochemical variability; consequently, they would be graded as severe C65 variants with either a mammals-only protein multiple sequence alignment or with our complete alignment through the deuterostomate Strongylocentrotus purpuratus. In contrast, the MRE11A rMS that described from a breast cancer case (p.Thr481Ile), as well as the rMS observed at the same position in a pair of ataxia-telangiectasia-like disease cases (p.Thr481Lys), score as severe C65 substitutions when evaluated with the mammals-only alignment but as likely innocuous C0 substitutions when evaluated with the evolutionarily deep alignment. Since the observation of a nonconservative rMS at MRE11A position Thr481 in a pair of ataxia-telangiectasia-like disease cases increases the odds that substitutions at this position are in fact pathogenic, it appears that using the evolutionarily deeper alignments is, for the MRN genes, counterproductive. On the other hand, the empirically determined allele frequency thresholds derived by combining older ATM, BRCA1, BRCA2, and CHEK2 case-control mutation screening data with EVS and 1000G data - found to be 0.1% for the three genes (ATM, BRCA1, and BRCA2) where inheritance of biallelic mutations is either embryonic lethal or causes a developmental phenotype that severely reduces reproductive fitness, and 0.32% for CHEK2 - provides a new tool to help with evaluation of the many rare variants observed in a case-control mutation screening study of candidate cancer susceptibility genes.
For BRCA1 and BRCA2, it is well established that a strong majority of pathogenic variants are, ultimately, protein-truncating variants. In contrast, case-control mutation screening of CHEK2 revealed an approximately equal contribution from T + SJVs and rMSs to the fraction of breast cancer attributable to rare variants in that gene, and a case-control mutation screening meta-analysis of ATM revealed that rMS in that gene may actually be responsible for a larger fraction of the breast cancer attributable to rare variants than are the T + SJVs [19, 20]. In the mutation screening data reported here, rare key functional domain missense substitutions in the MRN genes were more frequent (24 vs. 12 observations) than truncating variants and conferred a slightly higher OR (3.07 vs. 2.61) with a lower P value (0.029 vs. 0.14). These data are more congruent with the ATM/CHEK2 pattern than the BRCA1/2 pattern. Since there is not yet any efficient approach to clinically actionable classification of missense substitutions in these genes, these data point toward a clinical problem. When the MRN genes are mutation screened as part of a clinical panel-based cancer susceptibility gene sequencing test, a large fraction, if not the majority, of the genetic risk attributable to them will reside in rare missense substitutions that will initially be reported to clinical geneticists as unclassified variants.
The analytic strategy of treating the three genes as a single concatenated gene had one notable drawback: we are not able to ask whether variants in each of the three genes are best evaluated under the same analysis model. Thus an enormous amount of work, likely involving larger scale mutation screening efforts to gain more analytic precision, tests of segregation to examine penetrance and tumor spectrum, and perhaps development of functional assays to aid evaluation of rare missense substitutions, remains to be performed on with MRE11A, RAD50, and NBN.
Results reported here establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes and help to justify their inclusion on panel-based cancer susceptibility gene tests. Protein-truncating variants and rare missense substitutions falling in the key functional domains of these proteins appear to confer two- to three-fold increased risk of breast cancer. Like ATM and CHEK2, the spectrum of pathogenic variants in the MRN genes includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study. Given the relatively low frequency of likely pathogenic variants in the MRN genes, development of clinically applicable rare missense substitution classification models for these genes will require data from very large observational studies supplemented, in all likelihood, by carefully calibrated functional assays.
1000 genomes project
- Align-GVGD:
Align Grantham variation Grantham deviation
ataxia telangiectasia mutated
Breast Cancer Family Registry
Breast Cancer Information Core
- bp:
base pair
Breast Cancer 1 gene
BRCA1 and/or BRCA2
Breast Cancer 2 gene
checkpoint kinase 2
confidence interval
Canadian Institutes for Health Research
double-strand breaks
exome variant server
high-resolution melting curve analysis
International Agency for Research on Cancer
Institutional Review Board
Mendelian Inheritance in Man
meiotic recombination 11
- MRN complex:
MRE11-RAD50-NBN complex
United States National Cancer Institute
- NGRL Manchester:
UK National Reference Laboratory Manchester
United States National Institutes of Health
odds ratio
polymerase chain reaction
Phylogeny Inference Package
- PolyPhen:
polymorphism phenotyper
- rMS:
rare missense substitution
Sorting Intolerant from Tolerant
single-nucleotide polymorphism
- T- or M-Coffee:
Tree-based consistency objective function for alignment evaluation
truncating and splice junction variants
whole-genome amplified deoxyribonucleic acid
X-ray cross complemention group 2.
We wish to thank all participants in the BCFR for their contribution to the study. We also appreciate the support of J. McKay and the Genetic Cancer Susceptibility group at IARC.
Funding statement
This work was supported by the United States National Institutes of Health (NIH) National Cancer Institute (NCI) grant R01 CA121245, by the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer program, by the Government of Canada through Genome Canada and the Canadian Institutes of Health Research, and the Ministère de l'enseignement supérieur, de la recherche, de la science, et de la technologie du Québec through Génome Québec. The BCFR was supported by grant UM1 CA164920 from the NCI. The work also benefited from the Huntsman Cancer Institute's Bioinformatics Shared Resource, which is supported by NCI grant P30 CA042014. The content of this manuscript does not necessarily reflect the views or policies of the NCI or any of the collaborating centers in the BCFR, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government or the BCFR.
Authors' Affiliations
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- You Z, Chahwan C, Bailis J, Hunter T, Russell P: ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol Cell Biol. 2005, 25: 5363-5379.View ArticlePubMedPubMed CentralGoogle Scholar
- SIFT. [http://sift.jcvi.org/]
- PolyPhen-2. [http://genetics.bwh.harvard.edu/pph2/]
- di Masi A, Antoccia A: NBS1 heterozygosity and cancer risk. Curr Genomics. 2008, 9: 275-281.View ArticlePubMedPubMed CentralGoogle Scholar
- di Masi A, Viganotti M, Polticelli F, Ascenzi P, Tanzarella C, Antoccia A: The R215W mutation in NBS1 impairs gamma-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients. Biochem Biophys Res Commun. 2008, 369: 835-840.View ArticlePubMedGoogle Scholar
- Dzikiewicz-Krawczyk A, Mosor M, Januszkiewicz D, Nowak J: Impact of heterozygous c.657–661del, p.I171V and p.R215W mutations in NBN on nibrin functions. Mutagenesis. 2012, 27: 337-343.View ArticlePubMedGoogle Scholar
- Delia D, Piane M, Buscemi G, Savio C, Palmeri S, Lulli P, Carlessi L, Fontanella E, Chessa L: MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder. Hum Mol Genet. 2004, 13: 2155-2163.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Corporate events - Nash Conservatory at Kew
- Theatre style: 200
- Cabaret style: 80
- Standing reception: 200
- Dinner dance: 100
- Dinner: 120
- Day/evening hire 9am to 11pm.
- 9-hour period within the above times
- Additional hours charged as required
- 5 January to 22 December (excluding 10 February to 4 April 2014)
- Not available on public holiday dates throughout the year
- Monday to Friday exclusive use hire £4,500 + VAT
- Saturday to Sunday exclusive use hire £6,000 + VAT
- Not available for standalone hire on Friday to Sunday dates between April and October
Originally built in the grounds of Buckingham Palace the Nash Conservatory was moved brick-by-brick to Kew in 1836. The Nash Conservatory can accommodate a wide range of events, from presentations to product launches, corporate away days to drinks receptions, and much more.
With floor to ceiling windows on three sides this conservatory provides a wealth of natural daylight for your event.
The Nash Conservatory may be used for pre-dinner drinks before moving on to the Orangery for an evening dinner dance or perhaps as a lunch venue when using the Sir Joseph Banks Buiding for your conference.
Multiple venue hire packages
Bespoke venue hire packages may be arranged across any combination of Kew's portfolio of venues for guests who wish to utilise multiple event spaces within the Gardens.

subset: commoncrawl


text:
Greetings Guest!!Dismiss Notice
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(Player Event) ECL Season Four, Match 6 -- Monday 13 August, 8PM UK Time
Discussion in 'UO Europa' started by Aurelius, Aug 11, 2012.


text:
Video through photos fora Restaurant
- Status Closed
- Budget $30 - $250 USD
- Total Bids 31
Project Description
Dear all
We need a video in HD with the use of pictures from our restaurant
All the pictures are food related and we will add in the video some slogans along with the pictures
We need the video to be attractive, elegant and making people to want to taste our dishes
The duration will be 2-3 minutes maybe more
We need to see more works from you on videos, we are not interested on websites and other services.
If you do not send videos or if you send irrelevant materials you will be ignored
We need only serious bidders
Happy BiddingGet free quotes for a project like this
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- Set your budget and the timeframe
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- Forbes
- The New York Times
- Time
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- Times Online


text:
Joshua had attended upon Moses. He who was called to honour, had been long used to business. Our Lord Jesus took upon him the form of a servant. Joshua was trained up under command. Those are fittest to rule, who have learned to obey. The removal of useful men should quicken survivors to be the more diligent in doing good. Arise, go over Jordan. At this place and at this time the banks were overflowed. Joshua had no bridge or boats, and yet he must believe that God, having ordered
the people over, would open a way. (Jos 1:5-9)
Next commentary:
God promises to assist Joshua.
About this commentary:
Matthew Henry's Concise Commentary on the Bible is available in the Public Domain.
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text:
Dillion Harper - Wet Massage (2013) [FullHD 1080p]
Here you will find fresh porn videos, movies for every taste. You can download their by clicking on the link site file-sharing service Nitroflare, Uploaded, Gigapeta, Keep2Share.cc and many other.
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text:
John Carter Cash Net Worth: John Carter Cash is an American country singer-songwriter, author, and producer who has a net worth of $10 million. John Carter Cash was born in Nashville, Tennessee in March 1970. John is the only child of Johnny Cash and June Carter Cash. He has six half-siblings including Rosanne Cash, Carlene Carter, and Rosie Nix Adams. John started working with his mother as a music producer on her album Press On which won a Grammy in 1999. He worked under Rick Rubin as an Associate Producer on his father Johnny Cash's Grammy winning albums American III: Solitary Man and American IV: The Man Comes Around. John also produced his mother's album Wildwood Flower which won a Grammy Award for Best Traditional Folk album in 2003. He was nominated for three Grammy Awards for producing 2004's Unbroken Circle: The Musical Heritage of the Carter Family. He has also produced for Loretta Lynn, Brooks & Dunn, Elvis Costello, George Jones, Mavis Staples, Sheryl Crow, Kris Kristofferson, Emmylou Harris, Willie Nelson, and Vince Gill. Cash's debut album Bitter Harvest was released in 2003 and he also released the album The Family Secret. John has authored three children's books and his first novel Lupus Rex was published in 2013. He owns and operates Cash Productions LLC and the Cash Cabin Studio. In October 2014 Cash was arrested in a Canadian airport for stripping to his underwear.
John Carter Cash Net Worth
John Carter Cash
Record producer
Date of Birth
Mar 3, 1970 (46 years old)
Record producer, Film Producer, Singer-songwriter, Author, Songwriter, Music Producer, Actor
United States of America
More Singers
All net worths are calculated by applying a proprietary algorithm. The results are then fact checked and confirmed by a team of editors. We scan all publicly available data and resources and work diligently to ensure that our numbers are the most accurate and up to date net worth data you will find anywhere on the internet.


text:
Prompt used: Imagine person A of your otp upsetting person B's best friend by mistake. Person B then decides to punish person A by pulling them into a closet at work, undressing them, getting them as excited and aroused as possible, and then leaving them there unfinished and desperate for more.
Zoro was always a man of action, not really inclined to use many words, but he always thought before he spoke; that's why it was baffling when he simply... didn't. Think, that is. Not one to mull over trivial matters, he barely noticed that his words upset Nami and therefore the cook, too. In reality, they all knew she was a money grabbing witch, but pointing out in the middle of dinner that Luffy would be better off paying her for one night than putting up with her in a real long-term relationship wasn't something one might say to a friend. Or his boyfriend's best friend. Or any lady in Sanji's vicinity. Yet, Zoro did just that and continued with his life without a care in the world, oblivious of the upset ginger or the grinding of Sanji's teeth.
Sanji remained silent, sitting at the table with his friends and dumbass boyfriend, but his brain was working overtime to think of a suitable punishment for the marimo. Dinner continued, Luffy's cry for more food distracting everyone from Zoro's remark. The choice between an awkward silence and battling Luffy for their own food was an easy one.
Nami idly threatened to raise Zoro's debt, knowing full well that it would fall on deaf ears. Sanji did his best to coddle her and wiggle around her with treats, drinks or whatever else he could offer from the restaurant's stock to cheer her up, to no avail. The cook, however, didn't know of the word defeat and kept at it. He's been at it for hours before the group decided to call it a night. Or whatever you might call 3 in the morning.
They left without remorse, not bothered by leaving Sanji to do the cleanup, with Zoro as his sole help. The cleanup has always been a silent affair between the two of them and for the first time since they were dating, Sanji was welcoming the silence; it made the last planning that much easier.
The dining room of the restaurant was spotless within the hour, thus the two started on the dishes. Sanji washed while Zoro dried.
"Cook, something wrong?" the marimo asked, surprising him with the soft, almost caring voice. The moss-for-brains actually noticed something was wrong. Cute, but it won't work.
"No, marimo, just tired." the cook replied. He closed his eyes for good measure, not expecting two strong arms to make their way around his waist. Sanji didn't open his eyes as a warm breath tickled the shell of his ear, that deep voice grumbling "Too bad that you're tired." Sanji shivered; that voice did things to him he'd be too embarrassed to admit out loud.
After a quick flick of the tongue that Sanji couldn't even be sure really happened, Zoro slid his hands over the cook's abdomen and hipbones before removing them. He was back at the cook's side, drying dishes as if he hadn't turned Sanji hard beyond belief with those simple words. A slight blush crept to his face; damn shitty marimo getting him worked up like that.
Sanji's eyes shot open as an epiphany hit him. I can use this against him! Forgetting all previous plans, the cook decided to just improvise and it should work to his advantage. With a smirk on his face, Sanji washed faster. Zoro was confused by the sudden change of pace, but went with it nonetheless. The smirk on the cook's face was something he'd seen enough times already to know what was in store for him.
Sanji handed him the last dish and Zoro did his best to dry it as fast as possible while the cook drained the sink. Barely finished with wiping his hands, the cook found himself hoisted onto the kitchen island in the middle of the room by a pair of hands gripping his hips. The hands still on his hips, holding him down, while Zoro leaned close and kissed the startled cook. Their lips moving together in slow motions, the kiss still surprisingly chaste. Sanji placed the palms of his hands onto Zoro's abdomen, tracing his abs upwards, sliding over his firm chest and shoulders, down do his shoulder blades then finally gripping the thin fabric of Zoro's shirt. He pulled the marimo closer, spreading his legs a bit wider so they can be as close as possible.
While Sanji's hands remained buried in the fabric of Zoro's shirt, the swordsman's hands started to roam. The kiss deepening, moving of lips quicker with a swift tongue darting here and there from both sides and Zoro started pulling on Sanji's prissy shirt. He pulled it out of his trousers at the back, reluctant to separate their bodies for a trivial task such as removing a shirt, but did so nonetheless as he knew Sanji would stop their activities if the shirt got hurt in any way. He moved only slightly, leaving enough space for a dexterous hand to swiftly unbutton the damn thing.
Parting for air was necessary, but still a nuisance, yet Zoro found the silver lining and took the time to slide the shirt from Sanji's shoulders. Sanji's arms were still around Zoro's neck and there was barely any space between them, but his shoulders were now bare. Zoro placed an adoring kiss on each of them before Sanji slid his hands off of the other's shoulders and behind himself on the counter, allowing the shirt to slide painfully slowly down his arms. The marimo enjoyed the view he was given; a panting Sanji with his shirt pooling around his hips, shadows dancing on the bare chest as he tried to steady his breathing.
Zoro's hands flew to the cook's face, gently tracing his jaw line and the barely noticeable stubble; something so unusual for the blonde that the marimo couldn't help himself linger there for a second longer. Sanji took a deep shaky breath and Zoro took it as an invitation for further exploration, never mind the fact that he knew the cook's body as well as his own. He closed in on the blonde, pressing their lips together as his fingers trailed slowly down the sides of Sanji's neck, earning a satisfied moan before they settled on his collarbones. The kiss was so gentle, it was almost painful: as painful as the state of their arousals, but Zoro kept the pace as it was. For the time being, at least.
The marimo nibbled at the blonde's lips, feeling the body underneath him tremble when his fingers continued their trail southwards. Sanji was still trembling when the fingers reached his nipples and he gasped, breaking the kiss unintentionally to moan in pure ecstasy. Zoro used this chance to latch onto the soft skin of his lover's neck, sucking at a spot he knew would drive the other mad while his fingers circled around Sanji's hardening nipples.
Sanji's consciousness was lingering between pleasure and pain, the marimo was moving too slow and it was messing with his sanity. Feeling both of the marimo's hands and his teeth simultaneously move once more, the cook moaned again. He was aware that it's a lost battle and his arms gave out; his back hit the flat surface of the counter, the breath knocked out of him. Zoro instinctively followed as the cook's body fell, continuing his course of action. He smirked when he noticed Sanjis hands were constricted by the shirt he failed to completely take off. Filing it under future bondage possibilities, Zoro's fingers reached the soft material of Sanji's pants and the cook couldn't help but moan in relief.
Playtime was over, as far as Zoro was concerned; the outline of Sanji's cock more than obvious under him. He didn't waste time unzipping the bothersome pants and sliding them off in a swift motion that was so natural to him already, he could do it without the cook's notice.
"Oh...hell...yes..." Sanji moaned, making Zoro smirk.
A pair of blue boxers stood between the swordsman and his goal. The cook was shivering, panting at the thought of what was to come. Zoro's mouth ghosted over Sanji's arousal for a few moments, the smirk still in place, before he lowered his head that one last inch and licked the length from base to tip. The moan that tore through the cook's throat was like music to Zoro's ear as well as his own rock-hard member.
Finally deeming it enough teasing, Zoro lifted Sanji's hips and removed the last bit of clothing the cook still wore; the shirt was acting as a restraint and thus was of use to the marimo. Sanji hissed at his sudden complete nudity while Zoro simply smirked before licking another slow, painfully slow trail along Sanji's length. Sanji's breathing was uneven, his eyes screwed shut and his arms held in place on either side of his hips.
The cook was losing his mind, such attentiveness and patience was almost unheard of with the marimo. His moan echoed through the kitchen when Zoro finally took him in his mouth. Hands now still, resting on Sanji's hips to hold him down and he picked up the pace. His tongue was darting up and down, mixing saliva and precum that already soaked through Sanji's now discarded boxers. No way in hell would he be able to last long and the marimo damn well knew it. He took all of Sanji in his mouth and sucked, earning moan after moan from the cook. The cook's voice was strained, breathing haggard and loud, but he couldn't cover his mouth as he usually did and Zoro enjoyed that fact to the fullest.
Zoro sped up again; he could see the cook's hands clenching in the crumpled shirt and he knew it was a matter of seconds. Hollowing his cheeks one last time, he sucked at Sanji's tip and the cook came with a moan of Zoro's name; evidence that he truly and fully enjoyed the performance. Zoro swallowed all of it with a smirk at the overly responsive side the cook displayed.
If Zoro could come from looking at one image, it would have been the one before him; the cook lying naked on his back with his legs spread, arms bound by his sides, clenched into the crumpled fabric of what was once a prissy shirt, his chest shiny with sweat, face flushed and breathing heavy, but those blue eyes crystal clear and pointed right at him. He could feel some of Sanji's come dripping from the corner of his mouth, but didn't bother with wiping it off.
Sanji came to his senses faster than expected and sat back up, untangling his hands from the damn shirt. His hands cupped Zoro's cheeks, but instead of a kiss Zoro was expecting, Sanji licked at the remnants of his release and had cleaned his lover's face. It was Zoro's turn to flush and get flustered as Sanji took control of the situation. He made quick work of taking the other's shirt off and tossing it on the counter behind his back. Sanji pressed himself against Zoro's naked chest and slid from the counter, never breaking the contact between their naked skin. He could feel how aroused Zoro was and the hiss he released showed him just how far gone he was.
Sanji hadn't forgotten about his intention to punish the marimo for his previous blunder with Nami and he knew it was his only chance. He wriggled the tips of his fingers into Zoro's pants and pulled towards himself, catching the other's lips for an apologetic kiss. He pushed himself away from the counter and guided the marimo backwards with his own body, his fingers working on the zipper. Finally undone, Sanji's pale hands slid into the tight jeans and caressed the soft skin of Zoro's thighs, successfully sliding the pants off. They pooled around Zoro's ankles for a moment before he stepped out of them; when had he taken off his shoes and socks, Sanji didn't know. Zoro's black boxers, soaked from sweat and precum, followed soon after and disappeared somewhere on the kitchen floor.
It pained Sanji to do this in the restaurant's kitchen, but not as much as he'd expected. His right hand gripped Zoro's member, making him shudder, while the left one pressed against his chest and still pushed him backwards. Backwards towards the walk-in fridge, a fact unknown to the marimo. Sanji was pumping his lover slowly, knowing he wouldn't last too long anyway, but the punishment called for leaving his part of the business unfinished. He kissed his way from Zoro's mouth, over his flushed cheek to a sensitive ear, loving the moans the swordsman gave him after each lick and nibble of the earlobe before he detached his mouth just enough to speak.
"I want to try something new." his voice was low and husky, just the way Zoro liked it. It sent shivers down his spine, it made his insides tingle and his cock beg for release. Not to mention the choice of words was almost orgasmic to his ears; the cook was adamant to experiments in the bedroom department until now.
Dexterous pale fingers left Zoro's chest and reached behind him. They came to a stop and Sanji captured the other's lips in a searing kiss before Zoro felt a sudden hit of cold air on his back. Sanji's lips stayed on Zoro's as he pushed him into the fridge, forcefully disconnecting them eventually. Zoro blinked in confusion at his new surroundings and stared at the cook, expecting him to follow. Why he would want sex in a fridge was beyond the swordsman, but Sanji was always odd to say the least.
Sanji, on the other hand, simply muttered "For Nami, you bastard." and closed the heavy door. His voice held no bite to it and he felt a bit bad about leaving the marimo like that, but he knew the geezer would be up in 10 to 15 minutes to prepare for the day.
He swiftly moved across the kitchen, ignoring the angry shouts from the heavy door and pulled his boxers on. They were uncomfortably moist, but he had other things on his mind. Using the nearest rag, correctly Zoro's shirt, he cleaned the counter that held a surprisingly detailed imprint of his butt. Collecting all the discarded clothes from the most random places of the kitchen, he ran upstairs and straight to the bathroom. He decided on a shower; he just knew he'd feel bad about leaving Zoro until he made it up to the marimo so why not making it up to him right away.
Sanji washed himself quickly, wanting to be in bed by the time Zoro was let out. He dried himself even faster and ran to his room wrapped in a big fluffy towel. It was a dumb thing to do when you live with your dad, but his room wasn't that far from the bathroom so he decided to risk it. Upon entering his room, he threw the towel in what he hoped was the direction of his desk. He knew there would come a time when he'd regret what he was about to do, but he dropped that thought as soon as it formed in his mind and proceeded to his nightstand. Even in the dark, he knew where he was going and what he needed. The top draw was ajar so locating it wasn't a difficult feat, but it took some time to located the item he wanted; the bottle of scentless lube. Plopping on the bed, he uncapped the bottle and squirted some lube on his fingers, hoping the stupid marimo would appreciate what he had in mind. His hands, usually so steady, were trembling slightly; he had never prepared himself before and the feeling of his own lube coated fingers prodding his entrance was unusual to say the least. Still, he didn't falter and continued what he started with no sense of regret. He stopped for a fleeting second when he heard the distinct sound of the geezer's footsteps pass by his door, but continued when the sounds faded.
Zoro was freezing his balls off. It was definitely a first, but not a pleasant feeling in the slightest. Damn cook, just begging for trouble, pulling shit like that. He wasn't even sure what he did to Nami this time to invoke Sanji's wrath. It wasn't important, anyway, the cook could be a vindictive bastard when he wanted to be and Zoro should have known better. What he did know was that the cook would pay.
Noticing movement through the tiny window on the steel door, Zoro's temper flared and he waited for the damn cook to open the door. He waited for what seemed hours before he started banging on the door. It was sturdy, but the noise was loud enough to get anyone's attention. However, his anger dissipated as he was met with a tall chef's hat and a scowling face of a certain old cook that didn't care much for him. The old man held eye contact and his scowl only deepened as he opened to door to find the marimo completely naked. The situation was like none other before and he didn't know how to respond. Before he could, though, Zoro darted past him and up the stairs, muttering profanities about a swirly brow; Zeff could only imagine what had happened in his kitchen, but opted not to since it would probably scar him for the rest of his life. He also decided not to go upstairs for the time being for the same reason.
Zoro ran up the stairs and down the hall, easily finding his way for once, the rage dulling the cold he felt. He would make the cook pay, dammit!
He came to a halt in front of Sanji's door and took a deep breath before he barged in. The room was dark, but low squishing sounds and sharp breathing revealed the cook's presence. The origin of the sounds was unknown to the marimo, hence it confused him for a second. He slammed at the light switch by the door, ready to shout at his soon-to-be-dead boyfriend, but the shout never came. He almost did, however.
To his utter surprise, the cook was lying on his usually pristine bed, still naked, flushed and panting. His legs were spread to a degree he found to be very arousing with, to him the best part of the display, fingers pumping in and out of his ass. His cock was hard again and that did it for the swordsman. The cook opened his eyes and looked at Zoro without stopping what he was doing, offering only a smirk and a glance at his tongue as it darted over his parted lips. No words were needed, Zoro felt the chill drain from his body, replaced by what seemed as fire coursing through his veins. Hell, this was something new and most definitely arousing, he knew there would be no revenge in the near future if it meant more of this kinkier side of his cook.
"I thought you'd be cold..." Sanji broke the silence. "...so I thought I'd warm you up." he finished and lifted his other arm to motioned for Zoro to come closer.
Zoro hadn't hesitated for a second, slamming the door shut and almost pouncing on the cook. He moved between the cook's parted legs, gliding his hand over an exposed inner thigh in the process. Their lips connected in a kiss that was far rougher and needier than those they shared in the kitchen earlier. Zoro hadn't noticed when Sanji removed his fingers and coated them with more lube until he felt the cold substance on his already awakened member. He let Sanji coat him while he slid his hand under one of Sanji's knees and moved the pliant leg over his shoulder.
The second Sanji's hand left him, he positioned himself and entered the waiting cook. They moaned in unison, Zoro feeling like he hadn't been truly warm in years. Long bony fingers tangled in his hair was the only signal he needed; he pulled out almost all the way and slammed into the cook again.
Zoro didn't get his release as the cook did earlier so he was aware this would be a short affair, but he would make it count nonetheless. Forgetting his previous slow pace, Zoro moved the cook's hips and thrust into him hard. Sanji's pull on his hair told him he had hit the spot he wanted; the damn cook muffled his voice this time. No matter, Zoro was determined to get some screams of pleasure out of his lover and he clutched the cook's hips, thrusting in and moving the cook towards himself at the same time. The man in question was a shivering, panting mess, tangled in white sheets; nothing looked more appealing to Zoro at the moment. He lowered his head and planted a kiss on Sanji's lips, his thrusting continuing at the same pace.
The marimo was nearing his limit, the first waves of pleasure hitting him as he felt his climax burning a trail of pure ecstasy. He thrust harder and took a hold of Sanji's cock, knowing that his lover's climax would only bring him more pleasure. He pumped Sanji in sync with his thrusts, hitting his prostate dead on with every single one. The cook was almost chewing through his hand, trying to suppress a scream. His own finish was close at hand and, after an especially powerful thrust, he came on his abdomen with a scream of Zoro's name on his lips.
Zoro barely contained himself as long as he did, but with Sanji's scream and his already tight insides clenching even more around him, he let go. The pleasure took over him, the sight of Sanji's face fading from his eyes to be replaced with white, with stars, with everything and nothing at the same time. He was aware of his own pleasure and the soothing sound of Sanji's irregular breathing, the feel of the other's chest heaving under him.
"Still cold?" Sanji's voice purred in his ear, he could almost hear the smirk.
Zoro smiled, trying to catch his breath. God, he loved the vindictive bastard.
This is for TurtleFriedRice simply because she wrote smut for me. I didn't enjoy writing it at all. *whistles*


text:
Stay on top of what's going on in your neighborhood.
With fall just around the corner, the River Wards are alive again with meetings, trips, and community events. We've got the comprehensive list.
As the lazy days of summer dwindle down, the neighborhoods are buzzing yet again with events and activities. Star keep you up to date on them all.
Keep up to date on neighborhood events throughout the summer with our calendar.
Friday, May 11


text:
Hello Robiners,
I have a commercial friend in Western Australia who has asked me to try and
obtain some info. for him. He is also looking for somebody who will
either exchange or sell him some seed of Miniature or Dwarf Pelargoniums.
Info requested:
Are there any significant growers/hybridizers of 1) Paint Box series 2)
Rosebud flowered Ivy 'Geraniums' 3)Dwarf Zonals a)Gold Leaf types b) Paint
Box types
We hear of the huge market for Pelargoniums (incl so-called Geraniums) in
the USA,
what types are usually sold?? Are they Regals or Zonals? Are they named
varieties or bulked commercial types such as PAC or Fischers ?? Is there
any unsatisfied demand for the less common types of colored leaf,
miniature, deacon types, hybrid ivies or is the current demand adequately
met by current specialists such as Faye Brawner .
Are you experiencing the same situation in the US as we are where named
varieties are becoming not cost effective and therefore are being abandoned
by commercial growers in favour of the so-called bulk consumer/supermarket
types where there is an easier $ to be made???
I am concerned about the direction of modern society in the plant world and
although efforts are being made in some quarters to preserve species and
cultivars of significance there is little encouragement for hybridizers to
delve into the unknown and certainly they are a dying race in my Country,
how about yours?
Geraniaceae is all around the World


text:
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text:
Comment ...out of just about anything with carbon. (Score 1) 129
Soylent Graphene is people!!
the inventor hopes to replicate the sensations of breathing, saliva and taste in a future model.
I would probably focus more on making it FEEL and MOVE like a squishy tongue. That would be the killer enhancement. Still not sure I'd partake... but that'd be the logical direction to go... make it wet and squishy and more flexible.
"Achieving both liberty and security; protecting both transparency and confidentiality; protecting free expression while fostering tolerance and civility; are the three major challenges government as a whole is failing at today,"
but I fixed that for ya.
Another megabytes the dust.