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1.  Computational Screening and Molecular Dynamic Simulation of Breast Cancer Associated Deleterious Non-Synonymous Single Nucleotide Polymorphisms in TP53 Gene 
PLoS ONE  2014;9(8):e104242.
Breast cancer is one of the most common cancers among the women around the world. Several genes are known to be responsible for conferring the susceptibility to breast cancer. Among them, TP53 is one of the major genetic risk factor which is known to be mutated in many of the breast tumor types. TP53 mutations in breast cancer are known to be related to a poor prognosis and chemo resistance. This renders them as a promising molecular target for the treatment of breast cancer. In this study, we present a computational based screening and molecular dynamic simulation of breast cancer associated deleterious non-synonymous single nucleotide polymorphisms in TP53. We have predicted three deleterious coding non-synonymous single nucleotide polymorphisms rs11540654 (R110P), rs17849781 (P278A) and rs28934874 (P151T) in TP53 with a phenotype in breast tumors using computational tools SIFT, Polyphen-2 and MutDB. We have performed molecular dynamics simulations to study the structural and dynamic effects of these TP53 mutations in comparison to the wild-type protein. Results from our simulations revealed a detailed consequence of the mutations on the p53 DNA-binding core domain that may provide insight for therapeutic approaches in breast cancer.
PMCID: PMC4126775  PMID: 25105660
2.  The BARD1 Cys557Ser Variant and Breast Cancer Risk in Iceland 
PLoS Medicine  2006;3(7):e217.
Most, if not all, of the cellular functions of the BRCA1 protein are mediated through heterodimeric complexes composed of BRCA1 and a related protein, BARD1. Some breast-cancer-associated BRCA1 missense mutations disrupt the function of the BRCA1/BARD1 complex. It is therefore pertinent to determine whether variants of BARD1 confer susceptibility to breast cancer. Recently, a missense BARD1 variant, Cys557Ser, was reported to be at increased frequencies in breast cancer families. We investigated the role of the BARD1 Cys557Ser variant in a population-based cohort of 1,090 Icelandic patients with invasive breast cancer and 703 controls. We then used a computerized genealogy of the Icelandic population to study the relationships between the Cys557Ser variant and familial clustering of breast cancer.
Methods and Findings
The Cys557Ser allele was present at a frequency of 0.028 in patients with invasive breast cancer and 0.016 in controls (odds ratio [OR] = 1.82, 95% confidence interval [CI] 1.11–3.01, p = 0.014). The alleleic frequency was 0.037 in a high-predisposition group of cases defined by having a family history of breast cancer, early onset of breast cancer, or multiple primary breast cancers (OR = 2.41, 95% CI 1.22–4.75, p = 0.015). Carriers of the common Icelandic BRCA2 999del5 mutation were found to have their risk of breast cancer further increased if they also carried the BARD1 variant: the frequency of the BARD1 variant allele was 0.047 (OR = 3.11, 95% CI 1.16–8.40, p = 0.046) in 999del5 carriers with breast cancer. This suggests that the lifetime probability of a BARD1 Cys557Ser/BRCA2 999del5 double carrier developing breast cancer could approach certainty. Cys557Ser carriers, with or without the BRCA2 mutation, had an increased risk of subsequent primary breast tumors after the first breast cancer diagnosis compared to non-carriers. Lobular and medullary breast carcinomas were overrepresented amongst Cys557Ser carriers. We found that an excess of ancestors of contemporary carriers lived in a single county in the southeast of Iceland and that all carriers shared a SNP haplotype, which is suggestive of a founder event. Cys557Ser was found on the same SNP haplotype background in the HapMap Project CEPH sample of Utah residents.
Our findings suggest that BARD1 Cys557Ser is an ancient variant that confers risk of single and multiple primary breast cancers, and this risk extends to carriers of the BRCA2 999del5 mutation.
Editors' Summary
About 13% of women (one in eight women) will develop breast cancer during their lifetime, but many factors affect the likelihood of any individual woman developing this disease, for example, whether she has had children and at what age, when she started and stopped her periods, and her exposure to certain chemicals or radiation. She may also have inherited a defective gene that affects her risk of developing breast cancer. Some 5%–10% of all breast cancers are familial, or inherited. In 20% of these cases, the gene that is defective is BRCA1 or BRCA2. Inheriting a defective copy of one of these genes greatly increases a woman's risk of developing breast cancer, while researchers think that the other inherited genes that predispose to breast cancer—most of which have not been identified yet—have a much weaker effect. These are described as low-penetrance genes. Inheriting one such gene only slightly increases breast cancer risk; a woman has to inherit several to increase her lifetime risk of cancer significantly.
Why Was This Study Done?
It is important to identify these additional predisposing gene variants because they might provide insights into why breast cancer develops, how to prevent it, and how to treat it. To find low-penetrance genes, researchers do case–control association studies. They find a large group of women with breast cancer (cases) and a similar group of women without cancer (controls), and examine how often a specific gene variant occurs in the two groups. If the variant is found more often in the cases than in the controls, it might be a variant that increases a woman's risk of developing breast cancer.
What Did the Researchers Do and Find?
The researchers involved in this study recruited Icelandic women who had had breast cancer and unaffected women, and looked for a specific variant—the Cys557Ser allele—of a gene called BARD1. They chose BARD1 because the protein it encodes interacts with the protein encoded by BRCA1. Because defects in BRCA1 increase the risk of breast cancer, defects in an interacting protein might have a similar effect. In addition, the Cys557Ser allele has been implicated in breast cancer in other studies. The researchers found that the Cys557Ser allele was nearly twice as common in women with breast cancer as in control women. It was also more common (but not by much) in women who had a family history of breast cancer or who had developed breast cancer more than once. And having the Cys557Ser allele seemed to increase the already high risk of breast cancer in women who had a BRCA2 variant (known as BRCA2 999del5) that accounts for 40% of inherited breast cancer risk in Iceland.
What Do These Findings Mean?
These results indicate that inheriting the BARD1 Cys557Ser allele increases a woman's breast cancer risk but that she is unlikely to have a family history of the disease. Because carrying the Cys557Ser allele only slightly increases a woman's risk of breast cancer, for most women there is no clinical reason to test for this variant. Eventually, when all the low-penetrance genes that contribute to breast cancer risk have been identified, it might be helpful to screen women for the full set to determine whether they are at high risk of developing breast cancer. This will not happen for many years, however, since there might be tens or hundreds of these genes. For women who carry BRCA2 999del5, the situation might be different. It might be worth testing these women for the BARD1 Cys557Ser allele, the researchers explain, because the lifetime probability of developing breast cancer in women carrying both variants might approach 100%. This finding has clinical implications in terms of counseling and monitoring, as does the observation that Cys557Ser carriers have an increased risk of a second, independent breast cancer compared to non-carriers. However, all these findings need to be confirmed in other groups of patients before anyone is routinely tested for the BARD1 Cys557Ser allele.
Additional Information.
Please access these Web sites via the online version of this summary at
• MedlinePlus pages about breast cancer
• Information on breast cancer from the United States National Cancer Institute
• Information on inherited breast cancer from the United States National Human Genome Research Institute
• United States National Cancer Institute information on genetic testing for BRCA1 and BRCA2 variants
• GeneTests pages on the involvement of BRCA1 and BRCA2 in hereditary breast and ovarian cancer
• Cancer Research UK's page on breast cancer statistics
In a population-based cohort of 1090 Icelandic patients, a Cys557Ser missense variant of the BARD1 gene, which interacts with BRCA1, increased the risk of single and multiple primary breast cancers.
PMCID: PMC1479388  PMID: 16768547
3.  Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue 
X-ray crystallographic structures of four p53 core-domain variants were determined in order to gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53.
To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol−1 (15.1 kJ mol−1). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.
PMCID: PMC3792646  PMID: 24100332
p53; cancer mutation; suppressor mutation; rescue
4.  BRCA1 and BRCA2 mutations in central and southern Italian patients 
Breast Cancer Research : BCR  2000;2(4):307-310.
Protein truncation test (PTT) and single-strand conformation polymorphism (SSCP) assay were used to scan the BRCA1 and BRCA2 genes in 136 unrelated Italian breast/ovarian cancer patients. In the sample tested, BRCA1 and BRCA2 equally contributed to site-specific breast cancer patients who reported one to two breast cancer-affected first-/ second-degree relative(s) or who were diagnosed before age 40 years in the absence of a family history of breast/ovarian cancer. BRCA1 and BRCA2 mutations were mostly found in patients with disease diagnosis before and after age 50 years, respectively. Moreover, in cases with familial clustering of site-specific breast cancer, BRCA1 mostly accounted for tumours diagnosed before age 40 years and BRCA2 for tumours diagnosed after age 50 years. The BRCA1 and BRCA2 mutation spectrum was consistent with a lack of significant founder effects in the sample of patients studied.
Germline BRCA1 and BRCA2 mutations account for most hereditary breast/ovarian cancers and are associated with male breast cancer. Furthermore, constitutional mutations in these genes may occur in breast/ovarian cancer patients that do not meet stringent criteria of autosomal-dominant predisposition. The relevance of BRCA1 and BRCA2 mutations in such patients is still debated.
We sought to determine the impact of BRCA1 and BRCA2 mutations in a population of patients from central and southern Italy. We analyzed the BRCA1 and BRCA2 coding regions in 136 unrelated probands: 117 females with breast/ovarian cancer and 19 males with breast cancer. This population of patients was mostly representative of cases who are at risk for hereditary susceptibility, but who do not meet stringent criteria of autosomal-dominant predisposition.
Probands, subclassified as follows, were consecutively recruited depending on informed consent from patients attending breast cancer clinics in Rome and Naples. Selection criteria for females were as follows: breast cancer with breast cancer family history [one to two first-/second-degree relative(s), n = 55]; breast cancer diagnosed before age 40 years (no breast/ovarian cancer family history, n = 28); bilateral breast cancer (regardless of age and family history, n =10); breast cancer associated with gastrointestinal, pancreatic or uterine cancers [synchronous/metachronous or in first-degree relative(s), n = 9]; breast or ovarian cancer with family history of breast-ovarian/ovarian cancer (at least 1 first-/ second-degree relative, n = 10); and ovarian cancer with no breast/ovarian cancer family history (n = 5). Males with breast cancer were recruited regardless of age and family history. BRCA1 exon 11 and BRCA2 exons 10 and 11 were screened by PTT. Coding BRCA1 exons 2, 3, 5-10 and 12-24 and BRCA2 exons 2-9 and 12-27 were screened by SSCP. Primers are listed in Table 1. In 27 cases, analyzed by PTT along the entire BRCA1 coding sequence, BRCA1 SSCP analysis was limited to exons 2, 5, 20 and 24. Mutations were verified by sequence analysis on two independent blood samples.
Deleterious germline BRCA1/BRCA2 mutations were detected in 11 out of 136 cases (8%). Only three BRCA2 mutations were novel. One BRCA2 mutation recurred in two unrelated probands. Table 2 shows the mutations and data concerning carriers and their families. Table 3 shows correlations between BRCA1/BRCA2 mutations and sex, age at disease diagnosis and familial clustering of breast/ovarian cancer in the total patient population. Table 4 shows the proportions of BRCA1 and BRCA2 mutations in females with site-specific breast and breast-ovarian/ovarian cancer. Table 5 shows the frequency of BRCA1/BRCA2 mutations in males. BRCA1 and BRCA2 mutations, respectively, accounted for four out of 68 (6%) and one out of 68 (1%) cases diagnosed before age 50 years, and for one out of 68 (1%) and five out of 68 (7%) cases diagnosed after age 50 years. BRCA1 mutations were found in five out of 117 females (4%) and in none of 19 males (0%), and BRCA2 mutations were found in four out of 117 females (3%) and in two out of 19 males (10%). The proportions of BRCA1 and BRCA2 mutations coincided in site-specific female breast cancers (four out of 102; ie 4% each). BRCA1 and BRCA2 equally contributed to female breast cancers, with no familial clustering in those diagnosed before age 40 years (one out of 28; 4% each), and to female breast cancers, all ages, with familial clustering in one to two relatives (three out of 55; ie 5% each). In the latter subset of cases, BRCA1 mostly accounted for tumours diagnosed before age 40 years (two out of eight; 25%), and BRCA2 for tumours diagnosed after age 50 years (three out of 34; 9%). Regardless of family history, the respective contributions of BRCA1 and BRCA2 to site-specific female breast cancers diagnosed before age 40 years were 8% (three out of 36) and 3% (one out of 36). One BRCA1 mutation was detected among the 15 female probands from breast-ovarian/ovarian cancer families (7%). Among male breast cancers, BRCA2 mutations were identified in one out of five (20%) cases with family history and in one out of 14 (7%) apparently sporadic cases. No BRCA1 or BRCA2 mutations were found in female probands with nonfamilial bilateral breast cancer (10 cases) or in those with breast cancer associated with gastrointestinal, pancreatic or uterine cancers, synchronous/metachronous or in first-degree relative(s) (nine cases). These cases were all diagnosed after age 40 years.
Our results indicate a lack of relevant founder effects for BRCA1- and BRCA2-related disease in the sample of patients studied, which is consistent with other Italian studies and with ethnical and historical data. Overall, the contribution of BRCA1 and BRCA2 to breast/ovarian cancer in Italian patients appears to be less significant than in patients from communities with founder mutations. The present study is in agreement with direct estimates on other outbred populations, indicating that 7-10% of all female breast cancers that occur in patients aged under 40 years are due to BRCA1/BRCA2.
We found that BRCA1 and BRCA2 equally contributed to site-specific breast cancers who had one/two breast cancer-affected first-/second-degree relative(s) or who were diagnosed within age 40 years in the absence of family history. This is consistent with recent data that indicated that the respective frequencies of BRCA1 and BRCA2 mutations are comparable in early onset breast cancer. Considering the total population of patients analyzed here, however, BRCA1 and BRCA2 mutations were mostly found in cases with disease diagnosis before and after age 50 years, respectively. Moreover, in cases with familial clustering of site-specific breast cancer, BRCA1 mostly accounted for tumours diagnosed before age 40 years, and BRCA2 for tumours diagnosed after age 50 years. This is in agreement with a trend, which has been observed in other populations, for the proportion of cases with BRCA2 mutations to increase, and the proportion with mutations in BRCA1 to decrease, as the age at cancer onset increases.
As in other studies, the frequency of BRCA1/BRCA2 mutations taken together was lower than the estimated frequencies at comparable ages for all susceptibility alleles derived from the Contraceptive and Steroid Hormones (CASH) study. The discrepancy between direct data deriving from BRCA1/BRCA2 mutational analysis and CASH estimates could be due to several factors, including contribution of gene(s) other than BRCA1/BRCA2, differences between populations and relative insensitivity of mutational screening. Only BRCA1 mutations were found in breast/ovarian and site-specific ovarian cancer families. BRCA2, but not BRCA1 mutations were found in the male breast cancers. The overall proportion of males with BRCA2 mutations was high when compared with data from other studies on outbred populations, but was low compared with data from populations with founder effects.
The present results should be regarded as an approximation, because the following types of mutation are predicted to escape detection by the screening strategy used: mutations in noncoding regions; missense mutations within BRCA1 exon 11 and BRCA2 exons 10 and 11; large gene deletions; and mutations within the first and last 180 nucleotides of the amplicons analyzed by PTT.
PMCID: PMC13918  PMID: 11056688
BRCA1; BRCA2; breast; carcinoma; germline mutations; Italy
5.  Ensemble-Based Computational Approach Discriminates Functional Activity of p53 Cancer and Rescue Mutants 
PLoS Computational Biology  2011;7(10):e1002238.
The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r2 = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.
Author Summary
p53 is a tumor suppressor protein that controls a central apoptotic pathway (programmed cell death). Thus, it is the most-mutated gene in human cancers. Due to the marginal stability of p53, a single mutation can abolish p53 function (“cancer mutants”), while a second mutation (or several) can restore it (“rescue mutants”). Restoring p53 function is a promising therapeutic goal that has been strongly supported by recent experimental results on mice. Understanding of the effects of p53 cancer and rescue mutations would be helpful for designing drugs that are able to achieve the same goal. The challenge is that cancer and rescue mutations are distributed widely in the protein, and experimental testing of all possible combinations of mutations is not feasible. This paper describes a simple computational metric that reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.
PMCID: PMC3197647  PMID: 22028641
6.  EBNA3A Association with RBP-Jκ Down-Regulates c-myc and Epstein-Barr Virus-Transformed Lymphoblast Growth 
Journal of Virology  2003;77(2):999-1010.
Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jκ/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jκ, did not change EBNA3C association with RBP-Jκ or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jκ, decreased c-myc expression, and caused G0/G1 growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jκ-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jκ binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jκ binding domain. The dependence of EBNA3A effects on the core RBP-Jκ interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jκ association strongly implicates RBP-Jκ in c-myc promoter regulation.
PMCID: PMC140836  PMID: 12502816
7.  Haploinsufficiency for BRCA1 is associated with normal levels of DNA nucleotide excision repair in breast tissue and blood lymphocytes 
BMC Medical Genetics  2005;6:26.
Screening mammography has had a positive impact on breast cancer mortality but cannot detect all breast tumors. In a small study, we confirmed that low power magnetic resonance imaging (MRI) could identify mammographically undetectable tumors by applying it to a high risk population. Tumors detected by this new technology could have unique etiologies and/or presentations, and may represent an increasing proportion of clinical practice as new screening methods are validated and applied. A very important aspect of this etiology is genomic instability, which is associated with the loss of activity of the breast cancer-predisposing genes BRCA1 and BRCA2. In sporadic breast cancer, however, there is evidence for the involvement of a different pathway of DNA repair, nucleotide excision repair (NER), which remediates lesions that cause a distortion of the DNA helix, including DNA cross-links.
Case presentation
We describe a breast cancer patient with a mammographically undetectable stage I tumor identified in our MRI screening study. She was originally considered to be at high risk due to the familial occurrence of breast and other types of cancer, and after diagnosis was confirmed as a carrier of a Q1200X mutation in the BRCA1 gene. In vitro analysis of her normal breast tissue showed no differences in growth rate or differentiation potential from disease-free controls. Analysis of cultured blood lymphocyte and breast epithelial cell samples with the unscheduled DNA synthesis (UDS) assay revealed no deficiency in NER.
As new breast cancer screening methods become available and cost effective, patients such as this one will constitute an increasing proportion of the incident population, so it is important to determine whether they differ from current patients in any clinically important ways. Despite her status as a BRCA1 mutation carrier, and her mammographically dense breast tissue, we did not find increased cell proliferation or deficient differentiation potential in breast epithelial cells from this patient which might have contributed to her cancer susceptibility. Although NER deficiency has been demonstrated repeatedly in blood samples from sporadic breast cancer patients, analysis of blood cultured lymphocytes and breast epithelial cells for this patient proves definitively that heterozygosity for inactivation of BRCA1 does not intrinsically confer this type of genetic instability. These data suggest that the mechanism of genomic instability driving the carcinogenic process may be fundamentally different in hereditary and sporadic breast cancer, resulting in different genotoxic susceptibilities, oncogene mutations, and a different molecular pathogenesis.
PMCID: PMC1215484  PMID: 15955237
8.  N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance 
PLoS Medicine  2007;4(12):e335.
The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.
Methods and Findings
The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 × 10−12). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.
This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.
Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes.
Editors' Summary
In the 1980s, infection with the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), was a death sentence. Although the first antiretroviral drugs (compounds that block HIV's life cycle) were developed quickly, single antiretrovirals only transiently suppress HIV infection. HIV rapidly accumulates random changes (mutations) in its genetic material, some of which make it drug resistant. Nowadays, there are many different antiretrovirals. Some inhibit the viral protease, an enzyme used to assemble new viruses. Others block reverse transcriptase (RT), which makes replicates of the genes of the virus. Nucleoside/nucleotide RT inhibitors (NRTIs; for example, zidovudine—also called AZT—and lamivudine) and non-nucleoside RT inhibitors (NNRTIs; for example, nevirapine and efavirenz) interfere with the activity of RT by binding to different sites in its so-called “DNA polymerase domain,” the part of the enzyme that constructs copies of the viral genes. Highly active antiretroviral therapy (HAART), which was introduced in the mid 1990s, combines several antiretrovirals (usually a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) so that the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. When treated with HAART, HIV infection is usually a chronic, stable condition rather than a fatal disease.
Why Was This Study Done?
Unfortunately, HIV that is resistant to drugs still develops in some patients. To improve the prevention and management of drug resistance, a better understanding of the mutations that cause resistance is needed. Resistance to RT inhibitors usually involves mutations in the DNA polymerase domain that reduce the efficacy of NRTIs (including thymidine analogue mutations—also known as TAMs—and lamivudine-resistance mutations) and NNRTIs. Blood tests that detect these resistance mutations (genotype tests) have been used for several years to guide individualized selection of HIV drugs. Recently, however, mutations outside the DNA polymerase domain have also been implicated in resistance to RT inhibitors. In this study, the researchers have used data and samples collected since the mid 1990s by Canada's British Columbia Centre for Excellence in HIV/AIDS to investigate the clinical relevance of a mutation called N348I. This mutation changes an asparagine (a type of amino acid) to an isoleucine in a region of RT known as the connection domain. The researchers have also investigated how this mutation causes resistance to RT inhibitors in laboratory tests.
What Did the Researchers Do and Find?
The researchers analyzed the first two-thirds of the RT gene in viruses isolated from a large number of the Centre's patients. Virus carrying the N348I mutation was present in less than one in 100 patients whose HIV infection had never been treated, but in more than one in 10 treatment-experienced patients. The mutation appeared early in therapy, often in viruses that had TAMs, a lamivudine-resistance mutation called M184V/I, and/or NNRTI resistance mutations. Patients treated with zidovudine and nevirapine were 2.6 times more likely to have the N348I mutation than patients not treated with these drugs. Furthermore, the appearance of the N348I mutation often coincided with an increase in viral load, although other mutations that appeared at a similar time could have contributed to this increase. When the researchers introduced the N348I mutation into HIV growing in the laboratory, they found that it decreased the susceptibility of the virus to zidovudine and to NNRTIs.
What Do These Findings Mean?
These findings show that the treatment of patients with RT inhibitors can select a drug-resistant HIV variant that has a mutation outside the enzyme's DNA polymerase domain. Because this N348I mutation, which is commonly selected in vivo and has also been seen in other studies, confers resistance to two classes of RT inhibitors and can emerge early during therapy, it could have a large impact on patient responses to antiviral regimens that contain zidovudine and nevirapine. Although these findings do not show that the N348I mutation alone causes treatment failure, they may have implications for genotypic and phenotypic resistance testing, which is often used to guide treatment decisions. At present, genotype tests for resistance to RT inhibitors look for mutations only in the DNA polymerase domain of RT. This study is the first to demonstrate that it might be worth looking for the N348I mutation (and for other mutations outside the DNA polymerase domain) to improve the ability of genotypic and phenotypic resistance tests to predict treatment outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including links to fact sheets (in English, French, and Spanish) about antiretrovirals, and chapters explaining antiretroviral resistance testing
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including fact sheets on types of HIV drugs, drug resistance, and resistance tests (in English, Spanish, French, Portuguese, and Russian)
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on treatment (in English and Spanish)
AIDSinfo, a service of the US Department of Health and Human Services provides information for patients on HIV and its treatment
PMCID: PMC2100143  PMID: 18052601
9.  A constitutional de novo mutation in exon 8 of the p53 gene in a patient with multiple primary malignancies. 
British Journal of Cancer  1996;74(2):269-273.
We report a constitutional point mutation of codon 278 in exon 8 of the TP53 gene that has not yet been described as a germ-line mutation. A 52-year-old female developed multiple primary malignancies (liposarcoma, breast cancer, malignant histiocytoma, occult adenocarcinoma). The mutation found in her tumour and peripheral blood lymphocyte DNA is a cytosine to thymine transition at the second position of codon 278 resulting in an amino acid exchange from proline to leucine in the DNA-binding domain. Evaluation of the patient's family revealed that both of her sons were affected by the same mutation. Although the patient's mother had died already, we were able to demonstrate by polymorphic microsatellite analysis that the defective allele originated from the maternal side. As four brothers and one sister had inherited the same allele, which however was wild type, we were able to show that the mutation must have occurred in the germ cells of the patient's mother and that it may therefore be called de novo. This explains the lack of a high cancer incidence in the family history. All tumours tested showed positive immunohistochemical staining for p53. Loss of heterozygosity was found in five of seven tumours, one showing chromosome 17 monosomy.
PMCID: PMC2074589  PMID: 8688334
10.  Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach 
A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.
PMCID: PMC3905962  PMID: 18285836
BRCA1; BRIP1; CtIP; hereditary breast cancer; missense variants
11.  Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: or at
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis
The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa®) or erlotinib (Tarceva®) in patients with advanced non-small cell lung cancer (NSCLC).
Clinical Need: Target Population and Condition
With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib).
Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined.
The Technology: EGFR mutation testing
The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%.
Research Questions
In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?
What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?
What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?
What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting?
A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms:
Non-Small-Cell Lung Carcinoma
Epidermal Growth Factor Receptor
An automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
The inclusion criteria were as follows:
Population: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)
Procedure: EGFR mutation testing before treatment with gefitinib or erlotinib
Language: publication in English
Published health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract)
Outcomes: progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).
The exclusion criteria were as follows:
Studies lacking outcomes specific to those of interest
Studies focused on erlotinib maintenance therapy
Studies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drug
Grey literature, where relevant, was also reviewed.
Outcomes of Interest
ORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)
Quality of Evidence
The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria.
Summary of Findings
Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel.
In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). However, erlotinib did not confer a significant survival benefit compared with placebo in patients with EGFR mutations (HR, 0.55; 95%CI, 0.25-1.19) versus wild-type (HR, 0.74; 95%CI, 0.52-1.05). The interaction between EGFR mutation status and erlotinib use was not significant (P=0.47). The lack of significance could be attributable to a type II error since there was a low sample size that was available for subgroup analysis.
A series of phase II studies have examined the clinical effectiveness of erlotinib in patients known to have EGFR mutations. Evidence from these studies has consistently shown that erlotinib yields a very high ORR (typically 70% vs. 4%) and a prolonged PFS (9 months vs. 2 months) in patients with EGFR mutations compared with patients with wild-type EGFR. Although having a prolonged PFS and higher respond in EGFR mutated patients might be due to a better prognostic profile regardless of the treatment received. In the absence of a comparative treatment or placebo control group, it is difficult to determine if the observed differences in survival benefit in patients with EGFR mutation is attributed to prognostic or predictive value of EGFR mutation status.
Based on moderate quality of evidence, patients with locally advanced or metastatic NSCLC with adenocarcinoma histology being treated with gefitinib in the first-line setting are highly likely to benefit from gefitinib if they have EGFR mutations compared to those with wild-type EGFR. This advantage is reflected in improved PFS, ORR and QoL in patients with EGFR mutation who are being treated with gefitinib relative to patients treated with chemotherapy.
Based on low quality of evidence, in patients with locally advanced or metastatic NSCLC who are being treated with erlotinib, the identification of EGFR mutation status selects those who are most likely to benefit from erlotinib relative to patients treated with placebo in the second or third-line setting.
PMCID: PMC3377519  PMID: 23074402
12.  Rare key functional domain missense substitutions in MRE11A, RAD50, and NBN contribute to breast cancer susceptibility: results from a Breast Cancer Family Registry case-control mutation-screening study 
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.
PMCID: PMC4229874  PMID: 24894818
13.  BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations 
The Journal of Cell Biology  2001;155(6):911-922.
The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator–containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5′ mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3′ region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions.
PMCID: PMC2150890  PMID: 11739404
BRCA1; BRCT; chromatin unfolding; breast cancer; COBRA1
14.  “Similarity Trap” in protein-protein interactions could be carcinogenic: simulations of p53 core domain complexed with 53BP1 and BRCA1 BRCT domains 
Structure (London, England : 1993)  2006;14(12):1811-1821.
Similar binding sites often imply similar protein-protein interactions and similar functions; however, similar binding sites may also constitute traps for nonfunctional associations. How are similar sites distinguished to prevent misassociations? BRCT domain from breast cancer-susceptibility gene product BRCA1 is structurally similar to 53BP1-BRCT domain, yet with different binding behavior with p53 core domain. 53BP1-BRCT domain forms a stable complex with p53. In contrast, BRCA1-p53 interaction is weak or other mechanisms operate. To delineate the difference, we designed thirteen BRCA1-BRCT mutants, and computationally investigated the structural and stability changes compared to the experimental p53-53BP1 structure. Interestingly, of the thirteen, the two mutations which are cancerous and involve non-conserved residues are those that enforced p53 core domain binding with BRCA1-BRCT in a way similar to p53-53BP1 binding. Hence, falling into the “similarity trap” may disrupt normal BRCA1 and p53 functions. Our results illustrate how this trap is avoided in the native state.
PMCID: PMC2429992  PMID: 17161371
protein-protein interactions; BRCT domain; 53BP1 BRCT-p53 interactions; BRCA1-p53 interaction; protein binding sites
15.  The BRCA1-interacting protein, Abraxas, is required for genomic stability and tumor suppression 
Cell reports  2014;8(3):807-817.
Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C-terminus BRCT domains interact with multiple proteins and are required for BRCA1's tumor suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients.
PMCID: PMC4149256  PMID: 25066119
16.  Interrogation of the Protein-Protein Interactions between Human BRCA2 BRC Repeats and RAD51 Reveals Atomistic Determinants of Affinity 
PLoS Computational Biology  2011;7(7):e1002096.
The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.
Author Summary
The atomic scale interactions that occur at the interfaces between proteins are fundamental to all biological processes. One such critical interface is formed between the proteins, human BRCA2 and RAD51. BRCA2 binds to and delivers RAD51 to sites of DNA damage, where RAD51 mediates the error-free repair of double-stranded DNA breaks. Mutations in BRCA2 have been linked to breast cancer predisposition. Therefore, an accurate picture of the interactions between these two proteins is of great importance. BRCA2 interacts with RAD51 via eight “BRC repeats” that are similar, but not identical, in sequence. Due to lack of experimental structural information regarding the binding of seven of the eight BRC repeats to RAD51, it is unknown how subtle sequence variations in the repeats translate to measurable variations in their binding affinity. We have used a range of computational methods, firstly based on classical force fields, and secondly based on first principles quantum mechanical techniques whose computational cost scales linearly with the number of atoms, allowing us to perform calculations on the entire protein complex. This is the first study comparing all eight BRC repeats at the atomic scale and our results provide critical insights into the control of RAD51 by human BRCA2.
PMCID: PMC3136434  PMID: 21789034
17.  RAD51 and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study 
PLoS ONE  2012;7(12):e52374.
Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including BRCA1, BRCA2, TP53, ATM, BRIP1, CHEK2 and PALB2, about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. RAD51 plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, RAD51C, RAD51D, and XRCC2, have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the RAD51 gene. We thus postulated that RAD51 could harbor rare germline mutations that confer increased risk of breast cancer.
Methodology/Principal Findings
We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in ATM and CHEK2. In total, 12 distinct very rare or private variants were characterized in RAD51, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery.
Altogether, our data suggest that RAD51 tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility.
PMCID: PMC3531476  PMID: 23300655
18.  Structural effects of the L145Q, V157F, and R282W cancer-associated mutations in the p53 DNA-binding core domain† 
Biochemistry  2011;50(23):5345-5353.
The p53 tumor suppressor is a transcription factor involved in many important signaling pathways, such as apoptosis and cell-cycle arrest. In over half of human cancers, p53 function is compromised by a mutation in its gene. Mutations in the p53 DNA-binding core domain destabilize the structure and reduce DNA-binding activity. We performed molecular dynamics simulations at physiological temperature to study the structural and dynamic effects of the L145Q, V157F, and R282W cancer-associated mutations in comparison to the wild-type protein. While there were common regions of destabilization in the mutant simulations, structural changes particular to individual mutations were also observed. Significant backbone deviations of the H2 helix and S7–S8 loop were observed in all mutant simulations; the H2 helix binds to DNA. In addition, the L145Q and V157F mutations, which are located in the β-sandwich core of the domain, disrupted the β-sheet structure and the loop-sheet-helix motif. The R282W mutation caused distortion of the loop-sheet-helix motif, but otherwise this mutant was similar to the wild-type structure. The introduction of these mutations caused rearrangement of the DNA-binding surface, consistent with their reduced DNA-binding activity. The simulations reveal detailed effects of the mutations on the stability and dynamics of p53 that may provide insight for therapeutic approaches.
PMCID: PMC3117291  PMID: 21561095
p53 tumor suppressor; oncogenic mutation; molecular dynamics; protein structure
19.  Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay 
Human Molecular Genetics  2012;21(18):3993-4006.
Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.
PMCID: PMC3428152  PMID: 22678057
20.  Mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection. 
Journal of Clinical Investigation  1998;101(11):2364-2369.
IFN-gamma is critical in the immune response to mycobacterial infections, and deficits in IFN-gamma production and response have been associated with disseminated nontuberculous mycobacterial infections. Mutations in the IFN-gamma receptor ligand-binding chain (IFNgammaR1) have been shown to confer susceptibility to severe infection with nontuberculous mycobacteria. However, mutations in the IFN-gamma receptor signal-transducing chain (IFNgammaR2) have not been described. We describe a child with disseminated Mycobacterium fortuitum and M. avium complex infections and absent IFN-gamma signaling due to a mutation in the extracellular domain of IFNgammaR2. In vitro cytokine production by patient PBMCs showed 75% less PHA-induced IFN-gamma production than in normal cells, while patient PHA-induced TNF-alpha production was normal. The normal augmentation of TNF-alpha production when IFN-gamma was added to endotoxin was absent from patient cells. Expression of IFNgammaR1 was normal, but there was no phosphorylation of Stat1 in response to IFN-gamma stimulation. DNA sequence analysis of the gene for IFNgammaR2 showed a homozygous dinucleotide deletion at nucleotides 278 and 279, resulting in a premature stop codon in the protein extracellular domain. This novel gene defect associated with disseminated nontuberculous mycobacterial infection emphasizes the critical role that IFN-gamma plays in host defense against mycobacteria.
PMCID: PMC508825  PMID: 9616207
21.  HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells 
Breast Cancer Research  2005;7(6):R1058-R1079.
Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2.
Cell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays.
We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature.
HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.
PMCID: PMC1410754  PMID: 16457687
22.  A Galectin-3 Sequence Polymorphism Confers TRAIL Sensitivity to Human Breast Cancer Cells 
Cancer  2011;117(19):4375-4380.
A common polymorphism, rs4644, coding for Pro64 or His 64 of the carbohydrate-binding protein galectin-3, influences susceptibility of galectin-3 to cleavage by matrix metalloproteinases and is associated with breast cancer incidence. Since forced expression of galectin-3 in a galectin-3-null breast cancer cell line confers sensitivity to TNF-Related Apoptosis-inducing Ligand (TRAIL), we sought to determine whether the His64/Pro64 polymorphism of galectin-3 affects the sensitivity to TRAIL.
Genomic DNA of breast cell lines was analyzed for SNP rs4644, and cytotoxicity was determined with MTT assay.
When a collection of 9 breast cell lines that express galectin-3 was examined for LGALS3 genotype and sensitivity to doxorubicin and TRAIL, doxorubicin sensitivity was not related to LGALS3 genotype. In contrast, 0/5 cell lines that were homozygous for Pro64 galectin-3 were TRAIL sensitive, but 2/2 homozygous His 64 cell lines and 1/2 heterozygous cell lines were sensitive to TRAIL. Forced expression of galectin-3 of defined genotype in galectin-3 null cells was used to more directly test the effect of the P64H mutation on TRAIL sensitivity. High level expression of His64 galectin-3 rendered BT549 cells sensitive to TRAIL and resistant to doxorubicin, but cells expressing Pro64 galectin-3 remained TRAIL-resistant and doxorubicin sensitive.
These results indicate that the naturally occurring P64H mutation in galectin-3 increases sensitivity to death receptor-mediated apoptosis. The conclusion could be relevant to disparities in breast cancer outcomes across population groups, and could guide design of future clinical trials of TRAIL-based therapies.
PMCID: PMC3164935  PMID: 21446041
Galectin-3; Single Nucleotide Polymorphism; TNF-Related Apoptosis-Inducing Ligand; breast cancer; apoptosis
23.  Assessment of SLX4 Mutations in Hereditary Breast Cancers 
PLoS ONE  2013;8(6):e66961.
SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.
Methods and Results
To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so.
Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.
PMCID: PMC3694110  PMID: 23840564
24.  Selected Aspects of Molecular Diagnostics of Constitutional Alterations in BRCA1 and BRCA2 Genes Associated with Increased Risk of Breast Cancer in the Polish Population 
This study was undertaken to determine: 1) Type and prevalence of founder mutations BRCA1 and BRCA2 genes in Polish families with strong aggregation of breast and/or ovarian cancer. 2) Risk of breast and/or ovarian cancer depending on type of BRCA1 gene mutation. 3) Prevalence of BRCA1 mutation and of other alleles presumably linked with predisposition to breast cancer in unselected Polish patients with breast cancer. 4) Risk of breast cancer in patients with 5972C/T polymorphism that alters the BRCA2 protein structure.
Summary of the results
1. Among 66 families from several regions in Poland with a strong aggregation of breast/ovarian cancer, founder mutation of the BRCA1 gene were disclosed in 34 families and of the BRCA2 gene in on family. Altogether, seven different mutations were disclosed. Five mutations were found in at least two families in this group. The most frequent mutation was 5382insC (18 families), followed by C61G (7 families) and 4153delA (4 families). 2. Among 200 families representative for Poland with strong aggregation of breast/ovarian cancer, mutation of the BRCA1 gene were found in 122 families (61%) and of the BRCA2 gene in seven families (3,5%). 119 out of 122 mutations of the BRCA1 gene (97,5%) were repeatable. Three recurrent mutations of the BRCA1 gene (5382insC, C61G, 4153delA) characteristic for the Polish population were disclosed in 111 families representing 86% of all pathogenic sequences of this gene. 3. The risk of ovarian cancer in carriers of the three most frequent recurrent mutation of the BRCA1 gene in Poland is similar (OR 43.6 for 5382insC and 50 for 4153delA). The risk of breast cancer is significantly different for 4153delA (OR 1) and for other mutations (OR 10.9). 4. Among 2012 unselected breast cancers diagnosed in hospitals of nine Polish cities, mutations of the BRCA1 gene (5382insC, C61G, 4153delA) were disclosed in 2.9% patients. CHEK2 alternation (1100delC, IVS2+1G>A, I157T) was discovered in 8.1% and NBS1 mutation (657del5) in 0.8% of the patients. The changes were more frequent in the study than the control group. However, the risk of breast cancer was significantly higher for only three of them. Two changes, namely 5382insC and C61G of the BRCA1 gene revealed a high penetrance (OR 6.2 and 15.0, respectively), while I157T of the CHEK2 gene was associated with a low risk of breast cancer (OR 1.4). Mutations of the BRCA1, CHEK2 and NSB1 genes were significantly more frequent in patients with breast cancer diagnosed prior to 50 years of age. The mean age at diagnosis was 47.2 years for carriers of the BRCA1 mutation, 50.7 years for NBS1 and 54.2 for CHEK2. The mean age at diagnosis in the group of patients without any if the mutations described above was 56.1 years. When breast cancer patients with the diagnosis before and after 50 years of age were compared, the greatest difference in the frequency of mutation was revealed for the BRCA1 gene (5.5% vs 1.5%).
BRCA1 mutations were significantly more frequent I familial aggregates of the tumor (10.8%), but were also present in sporadic cases (1.8%). For the CHEK2 and NBS1 genes, there was no correlation between frequency and family history of cancer in probands. 5. A higher frequency of heterozygous carriers of 5972C/T polymorphism of the BRCA2 gene was demonstrated for breast cancer prior to 50 years of age (OR 1.4). the risk of breast cancer prior to 50 years of age was particularly high in 5972T/T homozygote (OR 4.7). This polymorphism was associated with breast cancer notable for intraductal growth.
1. Efficient molecular diagnostics of genetic predisposition to breast/ovarian cancer in Poland could be based on relatively simple tests disclosing some of the most frequent recurrent mutations of the BRCA1 gene. 2. The risk of breast cancer seems to be only slightly higher in carriers of some BRCA1 gene mutations. This finding should be taken into account during work on prevention schemes for carriers of the BRCA1 mutations. 3. 5382insC and C61G mutations of the BRCA1 gene are linked with high risk of breast cancer. Changes in the CHEK2 and NBS1 genes appear to be linked with a higher risk of breast cancers, particularly at young age. However, penetrance in this case is low. All patients with breast cancer should be tested for BRCA1 gene mutations because the percentage of mutations is also high in patients older than 50 years of age or without familiar aggregation of breast/ovarian cancer. 4. Polymorphic changes in the BRCA2 gene sequence previously regarded as non-pathogenic may nevertheless predispose, homozygotes in particular, to breast cancer. Apparently, the recessive character of these changes is responsible for the negative family history in most cases. The use of DNA tests is the only way to disclose increased risk of breast cancer in carriers of the 5972T/T mutation.
PMCID: PMC4177211  PMID: 20223018
breast cancer; ovarian cancer; inherited predisposition; BRCA1; BRCA2
25.  Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿  
Molecular and Cellular Biology  2010;30(14):3503-3518.
Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome.
PMCID: PMC2897548  PMID: 20479120

Results 1-25 (1019224)