# Assessing Candidate Gene nsSNPs for Phenotypic Differences in Double-Strand Break Repair Using Radiation-InducedγH2A.X Foci

Authors: Christina A. Markunas; David M. Umbach; Zongli Xu; Jack A. Taylor
Journal: Journal of Cancer Epidemiology
 (2008)

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## Abstract

Nonsynonymous SNPs (nsSNPs) in DNA repair genes may be important determinants of DNA damage and cancer risk. We applied a set of screening criteria to a large number of nsSNPs and selected a subset of SNPs that were likely candidates for phenotypic effects on DNA double-strand break repair (DSBR). In order to induce and follow DSBR, we exposed panels of cell lines to gamma irradiation and followed the formation and disappearance ofγH2A.X foci over time. All panels of cell lines showed significant increases in number, intensity, and area of foci at both the 1-hour and 3-hour time points. Twenty four hours following exposure, the number of foci returned to preexposure levels in all cell lines, whereas the size and intensity of foci remained significantly elevated. We saw no significant difference in γH2A.X foci between controls and any of the panels of cell lines representing the different nsSNPs.


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## Body

## 1. Introduction

Defects in double-strand
break repair (DSBR) can lead to genome instability and eventually cancer [1]. Several disease syndromes have rare gene
mutations that disrupt DSBR and result in phenotypes with increased cancer risk
including Ligase IV (LIG4) syndrome, severe combined immunodeficiency with sensitivity
to ionizing radiation (RS-SCID), Ataxia-telangiectasia (A-T), Nijmegen breakage
syndrome (NBS), and Fanconi anemia complementation group D1 (FANCD1) [2]. Additionally, a growing literature shows that
common polymorphisms in DSBR genes can be associated with increased, and in
some cases decreased, risk of cancer [3–8].Recent resequencing efforts by the Environmental Genome
Project [9] and others have greatly expanded the
list of known single nucleotide polymorphisms (SNPs) within DNA repair genes. However,
only a few of these SNPs have
been examined in association studies, and even fewer have been functionally
characterized in vitro. A number of in silico tools including Sorting Intolerant From Tolerant (SIFT)
(http://sift.jcvi.org/), Polymorphism Phenotyping (PolyPhen) (http://genetics.bwh.harvard.edu/pph/data/index.html),
and SNPs3D (http://www.snps3d.org/) can be used
to predict a SNP's effect on protein function, structure, or gene regulation, although
in vitro functional studies are ultimately required to confirm those predictions.DSBs can be
evaluated in vitro by the localization of a phosphorylated form of the
histone variant H2A.X. In response to a
DSB, histone H2A.X molecules become rapidly phosphorylated on serine 139 by a
member of the phosphoinositide (PI) 3-kinase family which includesATM, DNA-PK, and ATR [10–14]. These phosphorylated H2A.X molecules, termed
γH2A.X, can span up to 2 Mb of chromatin surrounding a DSB and can be
visualized microscopically as distinct foci after fluorescent antibody labeling [11, 12]. Not only can DSBs, and in turn H2A.X phosphorylation, be induced by
exogenous agents such as ionizing radiation but they can also occur
endogenously during DNA replication, recombination in mitosis and meiosis,
apoptosis, senescence, telomere shortening, and V(D)J recombination [11–13, 15]. Formation of γH2A.X foci is cell cycle-dependent
and is greater in
S- and G2/M-phase than in G1, reflecting both DSBs created during replication
and the increased quantity of DNA available in which DSBs may occur [16].Previous reports
have demonstrated a direct association between the number of DSBs and the
number ofγH2A.X foci [11, 17]. Several studies have also shown that the
kinetics of DSBR correlates with γH2A.X induction and clearance by phosphatases
at low-dose radiation [17–20]. Using ionizing radiation doses similar to those
used in our study, several groups have used γH2A.X foci formation and
disappearance over time to successfully detect DSBR defects in cells with known
mutations in DSBR genes, such as LIG4,  XRS-6, DNA-PK, and ATM [17–19, 21].We applied a set
of screening criteria and in silico prediction tools to identify a set of 5
nsSNPs that appeared likely to affect DSBR. Using gamma irradiation to induce
DSBs, we evaluated panels of HapMap and Environmental Genome Project (EGP)
normal human lymphoblastoid cell lines representing each of the SNPs and
followed the kinetics of induction ofγH2A.X foci and their persistence over
time.


## 2. Materials and Methods

### 2.1. Selection of Nssnps

Using dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP) (build 126), we compiled a list of
1455 nsSNPs within 149 genes known to be involved in DNA repair
[22]. Of these, 227 nsSNPs had minor alleles that
were found as homozygotes or heterozygotes in 5 or more HapMap or EGP cell lines and thus met our
minimum frequency requirement. Using PolyPhen [23], 49 of 227 nsSNPs were predicted to be “possibly
damaging” or “probably damaging” to protein function based on phylogenetic,
sequence, and structural changes induced by the SNP. Of these 49 nsSNPs, 22 were in genes known to
be involved in DSBR and, after further evaluation using SIFT classification [24], we selected 5 nsSNPs for
detailed functional analysis (Tables 1 and 2).Table 1
Selected nsSNPs and consequences based on in silico predictions.
GeneRS numberAA changePredicted consequence from in silico ProgramsPolyPhenSIFT OrthologSIFT homologPrediction of damageScore*PredictionScore†PredictionScore†PNKPrs3739186Y196NProbably2.025Tolerated0.26Tolerated0.25BRCA1rs1799950Q356RPossibly1.965Damaging0.04Damaging0.03LIG4rs1805388T9IPossibly1.521Damaging0.01Damaging0.04WRNrs1346044C1367RProbably2.327Tolerated0.06Tolerated0.10ATMrs1800057P1054RProbably2.621Damaging0Damaging0*PolyPhen
scores: ≤1.5  =  benign, >1.5and≤2  =  possibly
damaging, >2  =  probably damaging.†SIFT scores: >0.05  =  tolerated, ≤0.05  =  damaging.Table 2
Minor allele frequency for selected nsSNPs.
GenensSNPEGP MAFHapMap MAF(rs#)CEUHCBJPTYRIPNKPrs37391860.0170000.034BRCA1rs17999500.0120.042000LIG4rs18053880.1880.1670.2610.0560.158WRNrs13460440.1940.2330.0780.0680.15ATMrs18000570.0110.025000EGP: Environmental Genome ProjectMAF: Minor allele frequencyCEU: Utah residents with ancestry from northern and
western EuropeHCB: Han Chinese from Beijing, ChinaJPT: Japanese from Tokyo, JapanYRI: Yoruban from Ibadan, Nigeria.


### 2.2. Selection of Cell Lines

For each nsSNP, at least 5 Epstein-Barr-Virus-transformed lymphoblastoid cell lines were purchased from the HapMap or Polymorphism
Discovery Resource collections (for EGP) at Coriell Institute for Medical
Research (Camden, NJ, USA). Depending on
the allele frequency of the particular SNP, the cell lines were either
homozygous for the allele of interest (WRN,
LIG4) or heterozygous  if the allele was rare (PNKP, BRCA1, ATM). In a few
cases, cell lines that were homozygous for either the WRN or LIG4 variant
allele were also heterozygous for one of the other nsSNPs (PNKP, BRCA1, or ATM). In
order to establish a panel of control cell lines we identified all HapMap or EGP cell lines
that were homozygous for the common alleles at all 5 of the candidate
polymorphic sites. From these, we
selected 6 control cell lines (2 from each of the 3 ethnic groups in HapMap) after
applying an additional criterion to minimize sequence variation at other
polymorphic sites in the 5 genes of interest.


### 2.3. Cell Culture and Irradiation Conditions

Lymphoblastoid cell lines were grown at 37°C in 5% CO2 and maintained by tri-weekly subculture in RPMI-1640 medium with
L-glutamine (GIBCO, Carlsbad, Calif, USA), which was supplemented with 15%
fetal bovine serum (Gemini Bio-Products, West Sacramento, Calif, USA), and 1%
antibiotic/antimycotic (GIBCO, Carlsbad, Calif, USA). The day before irradiation, cells were seeded
at 3.5×105 cells/mL in T25 flasks (BD Falcon, Franklin Lakes, NJ,
USA). Cells were exposed to 1.5 Gy on
ice in 1 mL complete media while under constant rotation using a J. L. Sheperd
Model 431 137Cs irradiator at a dose rate of 0.77 Gy/min.


### 2.4.γh2a.x Immunofluorescence

Unexposed
control and irradiated cells were resuspended in warm, complete media, briefly
placed in a 37°C water bath, and transferred to a 37°C incubator to complete
the remainder of a 1 hour, 3 hours, or 24 hours incubation. Preliminary dose and time course experiments using
a control cell line (GM12154) were carried out by allowing cells
to adhere to poly-D-lysine (BD, Franklin
Lakes, NJ, USA) coated cover slips. All subsequent experiments, however, utilized poly-D-lysine-coated chamber slides instead. Cells were
allowed to adhere for 10 minutes at 4°C and then fixed in freshly prepared 4%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa, USA) for 15
minutes at room temperature. Cells were washed once in PBS, placed in methanol
at −20°C for 1 minute, permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, Mo,
USA) for 10 minutes, blocked in 3% BSA (Sigma, St. Louis, Mo, USA) for 30 minutes,
and then incubated for 1 hour with antiphospho histone H2A.X (Ser139), clone
JBW301 (1:1000; Upstate, Billerica, Mass, USA). At this point, cells were washed three times for 1 minute in 1% BSA,
incubated for 1 hour with Alexa 488 goat antimouse IgG (1:5000; Molecular
Probes, Carlsbad, Calif, USA), and then washed again three times for 1 minute
with 1% BSA. Next, cells were incubated
for 5 minutes with 20 nM 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, Calif, USA), washed three times
for 1 minute with PBS, and then permanently mounted using Fluoromount-G
(Southern Biotech, Birmingham, Ala, USA). Slides were stored in the dark at 4°C until microscope analysis.


### 2.5. Image Acquisition and Processing

Images
of cells were acquired at room temperature using a Zeiss Axioplan 2 fluorescent
microscope equipped with a JAI M1 HiRes charge-coupled device (CCD) camera,
Metafer v3.2 software (MetaSystems, Altlussheim, Germany), and a 40X objective
lens. The DAPI channel was used to identify DAPI stained cells, followed by
detection of Alexa 488 stainedγH2A.X foci using the FITC channel. Nine focus
planes were captured at 0.75 μm intervals for each cell using the FITC channel.
Automated image processing produced a composite image from both channels (Figure 1) by using settings recommended by the manufacturer.  Imaging operations were applied uniformly
across all slides and time points.(a)Representative cell irradiated with 1.5 Gy and incubated for 1
hour following exposure. Left panel: unadjusted, noncompressed image viewed
under the DAPI channel; middle panel: unadjusted, noncompressed image viewed
under the FITC channel; right panel: adjusted, composite image. The composite
image underwent a series of image processing operations including the use of
nonlinear algorithms and application of a counterstain mask. (b) Each of the four panels in this image
displays a different, representative cell from each time point in sequence from
left to right: preexposure, 1 hour, 3 hours, and 24 hours after 1.5 Gy gamma irradiation. All represent adjusted composite images.
(a)(b)For each captured nuclear image, we collected a total DAPI
intensity value in order to quantify the amount of DNA present in a cell. In order to compensate for DAPI intensity
fading over a course of image capture, we adjusted for fading following the
method of Böcker et al. [25].


### 2.6. Quantification and Measurements ofγh2a.x Foci

Approximately 200 cells were scored for each
time point/treatment combination (condition) within an experiment, each
condition was replicated within an experiment, and each experiment was
replicated on a separate day. Foci were
identified from captured images after applying a 20% intensity threshold to
minimize background. Automated measurements of the number, intensity, and area
(μm2) of γH2A.X foci were made using
the Metafer v3.2 system (MetaSystems, Altlussheim, Germany). Using lower intensity
thresholds or adding area restrictions for size of foci did not substantively
alter the results (data not shown).


### 2.7. Cell Cycle Analysis

Cells were fixed with cold 70% ethanol and placed at −20°C
until flow analysis. Cells were washed once with PBS, incubated at room
temperature for 30 minutes with 0.5% Triton X-100, resuspended in propidium
iodide solution (5 μg/mL propidium iodide (Invitrogen, Carlsbad, Calif, USA)  +  10 u/mL RNase (Promega, Madison, Wis, USA)), and incubated for 30 minutes in
the dark before being processed using a Becton-Dickinson (BD) FACSort
Flow Cytometer and analyzed using both CellQuest (BD, Franklin Lakes, NJ, USA)
and Modfit software (Verity Software, Topsham, Me, USA).


### 2.8. Statistical Analysis

The
preliminary dose-response and time-response data on the number ofγH2A.X foci
were analyzed as follows: the mean of the two replicate slides was calculated
for each of the two experiments, and the mean of these two means and its standard error were then determined. To
assess the dose-response trend, we used linear regression methods.For
our primary analyses of the effects of nsSNPs on DSBR, we used mixed-model
regression techniques [26]. We measured three features
of γH2A.X foci (namely, number, intensity, and
size) at each of four time points (namely, preexposure, 1-, 3-, and 24-hour
postexposure (denoted T0, T1, T3, and T24, resp.)). For each feature, we used these measurements
to derive three response variables that compared time points. Each response
variable was a ratio representing fold changes in response between time
points: T1/T0 indicative of induction of
damage, T3/T1 indicative of short-term repair/persistence, and T24/T1
indicative of longer-term repair/persistence. These response variables were
assessed for each cell line in at least two replicated experiments on different
days. Although capacity restricted us to run at most nine cell lines in any
single experiment, generally each experiment included controls represented by
two or more cell lines and four or five nsSNPs represented by a single cell
line each. Our modeling approach involved fitting a separate multivariable
regression for each response variable. To better meet the normal-distribution
assumptions implicit in our statistical analysis, we used the base-2 logarithms of these ratio
variables when fitting models. Mixed models involve both a model for the mean
response and a model for the variation in response. The regression model for
the mean response included an intercept and five predictors, namely, the number
of copies of the variant (minor) allele for each of the five nsSNPs (control
cell lines had zero copies of all variants). The regression coefficients in
this model measure the change in mean response associated with an additional
copy of the variant allele at the given locus. This regression approach allowed
us to accommodate cell lines that carried variant alleles at more than one
nsSNP as well as cell lines that were either hetero- or homozygous for the
variant allele. It also allowed us to estimate the geometric mean response that
a cell line homozygous for any of the variants under investigation would have.
Our model for the variation in response accounted for several distinct sources
of variation and the correlations that they induce: among different cell lines
with the same genotype, among experiments, and among replicates of a given cell
line within an experiment.


## 2.1. Selection of Nssnps

Using dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP) (build 126), we compiled a list of
1455 nsSNPs within 149 genes known to be involved in DNA repair
[22]. Of these, 227 nsSNPs had minor alleles that
were found as homozygotes or heterozygotes in 5 or more HapMap or EGP cell lines and thus met our
minimum frequency requirement. Using PolyPhen [23], 49 of 227 nsSNPs were predicted to be “possibly
damaging” or “probably damaging” to protein function based on phylogenetic,
sequence, and structural changes induced by the SNP. Of these 49 nsSNPs, 22 were in genes known to
be involved in DSBR and, after further evaluation using SIFT classification [24], we selected 5 nsSNPs for
detailed functional analysis (Tables 1 and 2).Table 1
Selected nsSNPs and consequences based on in silico predictions.
GeneRS numberAA changePredicted consequence from in silico ProgramsPolyPhenSIFT OrthologSIFT homologPrediction of damageScore*PredictionScore†PredictionScore†PNKPrs3739186Y196NProbably2.025Tolerated0.26Tolerated0.25BRCA1rs1799950Q356RPossibly1.965Damaging0.04Damaging0.03LIG4rs1805388T9IPossibly1.521Damaging0.01Damaging0.04WRNrs1346044C1367RProbably2.327Tolerated0.06Tolerated0.10ATMrs1800057P1054RProbably2.621Damaging0Damaging0*PolyPhen
scores: ≤1.5  =  benign, >1.5and≤2  =  possibly
damaging, >2  =  probably damaging.†SIFT scores: >0.05  =  tolerated, ≤0.05  =  damaging.Table 2
Minor allele frequency for selected nsSNPs.
GenensSNPEGP MAFHapMap MAF(rs#)CEUHCBJPTYRIPNKPrs37391860.0170000.034BRCA1rs17999500.0120.042000LIG4rs18053880.1880.1670.2610.0560.158WRNrs13460440.1940.2330.0780.0680.15ATMrs18000570.0110.025000EGP: Environmental Genome ProjectMAF: Minor allele frequencyCEU: Utah residents with ancestry from northern and
western EuropeHCB: Han Chinese from Beijing, ChinaJPT: Japanese from Tokyo, JapanYRI: Yoruban from Ibadan, Nigeria.


## 2.2. Selection of Cell Lines

For each nsSNP, at least 5 Epstein-Barr-Virus-transformed lymphoblastoid cell lines were purchased from the HapMap or Polymorphism
Discovery Resource collections (for EGP) at Coriell Institute for Medical
Research (Camden, NJ, USA). Depending on
the allele frequency of the particular SNP, the cell lines were either
homozygous for the allele of interest (WRN,
LIG4) or heterozygous  if the allele was rare (PNKP, BRCA1, ATM). In a few
cases, cell lines that were homozygous for either the WRN or LIG4 variant
allele were also heterozygous for one of the other nsSNPs (PNKP, BRCA1, or ATM). In
order to establish a panel of control cell lines we identified all HapMap or EGP cell lines
that were homozygous for the common alleles at all 5 of the candidate
polymorphic sites. From these, we
selected 6 control cell lines (2 from each of the 3 ethnic groups in HapMap) after
applying an additional criterion to minimize sequence variation at other
polymorphic sites in the 5 genes of interest.


## 2.3. Cell Culture and Irradiation Conditions

Lymphoblastoid cell lines were grown at 37°C in 5% CO2 and maintained by tri-weekly subculture in RPMI-1640 medium with
L-glutamine (GIBCO, Carlsbad, Calif, USA), which was supplemented with 15%
fetal bovine serum (Gemini Bio-Products, West Sacramento, Calif, USA), and 1%
antibiotic/antimycotic (GIBCO, Carlsbad, Calif, USA). The day before irradiation, cells were seeded
at 3.5×105 cells/mL in T25 flasks (BD Falcon, Franklin Lakes, NJ,
USA). Cells were exposed to 1.5 Gy on
ice in 1 mL complete media while under constant rotation using a J. L. Sheperd
Model 431 137Cs irradiator at a dose rate of 0.77 Gy/min.


## 2.4.γh2a.x Immunofluorescence

Unexposed
control and irradiated cells were resuspended in warm, complete media, briefly
placed in a 37°C water bath, and transferred to a 37°C incubator to complete
the remainder of a 1 hour, 3 hours, or 24 hours incubation. Preliminary dose and time course experiments using
a control cell line (GM12154) were carried out by allowing cells
to adhere to poly-D-lysine (BD, Franklin
Lakes, NJ, USA) coated cover slips. All subsequent experiments, however, utilized poly-D-lysine-coated chamber slides instead. Cells were
allowed to adhere for 10 minutes at 4°C and then fixed in freshly prepared 4%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa, USA) for 15
minutes at room temperature. Cells were washed once in PBS, placed in methanol
at −20°C for 1 minute, permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, Mo,
USA) for 10 minutes, blocked in 3% BSA (Sigma, St. Louis, Mo, USA) for 30 minutes,
and then incubated for 1 hour with antiphospho histone H2A.X (Ser139), clone
JBW301 (1:1000; Upstate, Billerica, Mass, USA). At this point, cells were washed three times for 1 minute in 1% BSA,
incubated for 1 hour with Alexa 488 goat antimouse IgG (1:5000; Molecular
Probes, Carlsbad, Calif, USA), and then washed again three times for 1 minute
with 1% BSA. Next, cells were incubated
for 5 minutes with 20 nM 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, Calif, USA), washed three times
for 1 minute with PBS, and then permanently mounted using Fluoromount-G
(Southern Biotech, Birmingham, Ala, USA). Slides were stored in the dark at 4°C until microscope analysis.


## 2.5. Image Acquisition and Processing

Images
of cells were acquired at room temperature using a Zeiss Axioplan 2 fluorescent
microscope equipped with a JAI M1 HiRes charge-coupled device (CCD) camera,
Metafer v3.2 software (MetaSystems, Altlussheim, Germany), and a 40X objective
lens. The DAPI channel was used to identify DAPI stained cells, followed by
detection of Alexa 488 stainedγH2A.X foci using the FITC channel. Nine focus
planes were captured at 0.75 μm intervals for each cell using the FITC channel.
Automated image processing produced a composite image from both channels (Figure 1) by using settings recommended by the manufacturer.  Imaging operations were applied uniformly
across all slides and time points.(a)Representative cell irradiated with 1.5 Gy and incubated for 1
hour following exposure. Left panel: unadjusted, noncompressed image viewed
under the DAPI channel; middle panel: unadjusted, noncompressed image viewed
under the FITC channel; right panel: adjusted, composite image. The composite
image underwent a series of image processing operations including the use of
nonlinear algorithms and application of a counterstain mask. (b) Each of the four panels in this image
displays a different, representative cell from each time point in sequence from
left to right: preexposure, 1 hour, 3 hours, and 24 hours after 1.5 Gy gamma irradiation. All represent adjusted composite images.
(a)(b)For each captured nuclear image, we collected a total DAPI
intensity value in order to quantify the amount of DNA present in a cell. In order to compensate for DAPI intensity
fading over a course of image capture, we adjusted for fading following the
method of Böcker et al. [25].


## 2.6. Quantification and Measurements ofγh2a.x Foci

Approximately 200 cells were scored for each
time point/treatment combination (condition) within an experiment, each
condition was replicated within an experiment, and each experiment was
replicated on a separate day. Foci were
identified from captured images after applying a 20% intensity threshold to
minimize background. Automated measurements of the number, intensity, and area
(μm2) of γH2A.X foci were made using
the Metafer v3.2 system (MetaSystems, Altlussheim, Germany). Using lower intensity
thresholds or adding area restrictions for size of foci did not substantively
alter the results (data not shown).


## 2.7. Cell Cycle Analysis

Cells were fixed with cold 70% ethanol and placed at −20°C
until flow analysis. Cells were washed once with PBS, incubated at room
temperature for 30 minutes with 0.5% Triton X-100, resuspended in propidium
iodide solution (5 μg/mL propidium iodide (Invitrogen, Carlsbad, Calif, USA)  +  10 u/mL RNase (Promega, Madison, Wis, USA)), and incubated for 30 minutes in
the dark before being processed using a Becton-Dickinson (BD) FACSort
Flow Cytometer and analyzed using both CellQuest (BD, Franklin Lakes, NJ, USA)
and Modfit software (Verity Software, Topsham, Me, USA).


## 2.8. Statistical Analysis

The
preliminary dose-response and time-response data on the number ofγH2A.X foci
were analyzed as follows: the mean of the two replicate slides was calculated
for each of the two experiments, and the mean of these two means and its standard error were then determined. To
assess the dose-response trend, we used linear regression methods.For
our primary analyses of the effects of nsSNPs on DSBR, we used mixed-model
regression techniques [26]. We measured three features
of γH2A.X foci (namely, number, intensity, and
size) at each of four time points (namely, preexposure, 1-, 3-, and 24-hour
postexposure (denoted T0, T1, T3, and T24, resp.)). For each feature, we used these measurements
to derive three response variables that compared time points. Each response
variable was a ratio representing fold changes in response between time
points: T1/T0 indicative of induction of
damage, T3/T1 indicative of short-term repair/persistence, and T24/T1
indicative of longer-term repair/persistence. These response variables were
assessed for each cell line in at least two replicated experiments on different
days. Although capacity restricted us to run at most nine cell lines in any
single experiment, generally each experiment included controls represented by
two or more cell lines and four or five nsSNPs represented by a single cell
line each. Our modeling approach involved fitting a separate multivariable
regression for each response variable. To better meet the normal-distribution
assumptions implicit in our statistical analysis, we used the base-2 logarithms of these ratio
variables when fitting models. Mixed models involve both a model for the mean
response and a model for the variation in response. The regression model for
the mean response included an intercept and five predictors, namely, the number
of copies of the variant (minor) allele for each of the five nsSNPs (control
cell lines had zero copies of all variants). The regression coefficients in
this model measure the change in mean response associated with an additional
copy of the variant allele at the given locus. This regression approach allowed
us to accommodate cell lines that carried variant alleles at more than one
nsSNP as well as cell lines that were either hetero- or homozygous for the
variant allele. It also allowed us to estimate the geometric mean response that
a cell line homozygous for any of the variants under investigation would have.
Our model for the variation in response accounted for several distinct sources
of variation and the correlations that they induce: among different cell lines
with the same genotype, among experiments, and among replicates of a given cell
line within an experiment.


## 3. Results

### 3.1. Dose Response and Time Course for The Formation of Foci Following Gamma Irradiation

In preliminary experiments using a control
cell line (GM12154) with cells affixed to coated cover slips, we found a linear
dose-response relationship (R2  =  0.99) in the mean number of
γH2A.X foci per cell 0.5 hour after
exposures of up to 1.5 Gy of gamma irradiation, with the highest dose producing
a tripling in the number of foci per cell compared to unexposed cells (Figure 2(a)).
Subsequent time course experiments showed that the number of gamma-induced foci
reached a maximum at 0.5 hour following exposure to 1.5 Gy, decreased by ~50%
from this maximum by 3 hours, and returned to preexposure levels by 24 hours (Figure 2(b)). Allowing cells to affix to coated chamber slides instead of
coated cover slips resulted in modest but similar reductions in the mean number
of foci in both exposed and unexposed cells and in reduced experimental
variability, so we utilized chambered slides for all subsequent experiments. Based on preliminary
flow cytometry data, we estimated that about 60% of cells were in G0/G1 and
thus we used adjusted DAPI intensity as a measure of DNA content to exclude the
40% of cells in G2/M or S-phase for all analyses (data not shown).Preliminary dose- and time-response patterns
based on control cell line GM12154 for the mean number ofγH2A.X foci following
gamma irradiation. Two independent experiments, each containing two replicates
(~200 cells each), were used for the analyses. (a) Linear dose response at 0.5
hour following gamma irradiation (R2  =  0.99). Error bars represent ±1 SEM for two
experiments. (b) Non-linear time response to 1.5 Gy gamma irradiation. The time
point, NT, represents preexposure cells. Error bars represent  ±1 SEM for two
experiments.
(a)(b)We compared groups
of cell lines representing each of the DNA repair gene polymorphisms to the
group of cell lines used as a control (Figure3). All groups of cell lines exhibited
similar time-course response patterns for the mean number of γH2A.X foci per cell before and following
exposure to 1.5 Gy of gamma irradiation (Figure 3(a)). The mean number of foci increased about fourfold from before
exposure to 1 hour postexposure in all of the groups of cell lines. Average
number of foci remained more than twofold elevated in all groups of cell lines 3 hours after
exposure, but all groups returned to near-baseline levels by 24 hours.Summaries of gene-(nsSNP-)specific
temporal response patterns following 1.5 Gy gamma irradiation for three different response measures. Time points are
NT/preexposure, 1 hour, 3 hours, and 24 hours following irradiation. Error bars
represent 95% confidence limits.
(a) Mean number of foci. (b) Mean area of foci. (c) Mean intensity of foci.
(a)(b)(c)Although all cell lines appear by inspection
to have similar induction and disappearance ofγH2A.X foci, we statistically tested whether any
of the 5 nsSNPs differed from controls using a mixed-model regression
analysis with the number of copies of the variant allele at each nsSNP as
predictors. This approach can
accommodate cell lines that may be heterozygous or homozygous at a locus or
that may have variants at more than one locus. We considered three response
variables constructed as ratios of the number of foci at different time points:
induction of foci at 1hour compared to preexposure (T1/T0), the repair of this
damage over a 3-hour time course (T3/T1), and the persistence of damage at 24 hours
compared to preexposure levels (T24/T0). All groups of cell lines had similar estimates for these ratios, and we
saw no evidence that any group of cell lines differed from controls (Table 3).Table 3
Comparison of the number of foci at various time points (T0, before irradiation; T1, T3, T24, respectively, 1 hour, 3 hours, 24 hours, following irradiation.)
GenensSNPRatios of geometric means†T1/T0T3/T1T24/T0PNKPrs37391864.650.671.25BRCA1rs17999504.510.651.09LIG4rs18053883.940.641.08WRNrs13460443.830.641.11ATMrs18000574.200.621.31CONTROLS4.100.641.16†None of the ratios for any cell line panel representing
SNPs was statistically
significantly different from the corresponding ratio for controls. (P>.05, t-tests comparing coefficients from a mixed model regression
fitted separately to the base-2 logarithm of each ratio).In
addition to measuring the number of foci within cells, we also measured the
size and intensity of foci, again concentrating on ratio responses analogous to
those mentioned earlier. By one hour after exposure, the mean size of
individual foci increased by more than 75% compared to preexposure levels in
all groups of cell lines and remained elevated and virtually unchanged at 3 hours
following exposure (Figure2(b)). Even
24 hours after exposure, when the average number of foci per cell had returned
to near preexposure levels, the average size of the foci remained significantly
larger than that of cells before exposure (P<.0001 for all
groups, P=.0003 for PNKP). One
hour following exposure, mean intensity of foci increased more than twofold
over preexposure levels, and it decreased only slightly from the 1 hour levels
at 3 hours (Figure 3(c)). Mean intensity
remained significantly elevated after 24 hours in all groups of cell lines
except those with ATM and PNKP polymorphisms, although even these
two had elevated intensities at 24 hours.


## 3.1. Dose Response and Time Course for The Formation of Foci Following Gamma Irradiation

In preliminary experiments using a control
cell line (GM12154) with cells affixed to coated cover slips, we found a linear
dose-response relationship (R2  =  0.99) in the mean number of
γH2A.X foci per cell 0.5 hour after
exposures of up to 1.5 Gy of gamma irradiation, with the highest dose producing
a tripling in the number of foci per cell compared to unexposed cells (Figure 2(a)).
Subsequent time course experiments showed that the number of gamma-induced foci
reached a maximum at 0.5 hour following exposure to 1.5 Gy, decreased by ~50%
from this maximum by 3 hours, and returned to preexposure levels by 24 hours (Figure 2(b)). Allowing cells to affix to coated chamber slides instead of
coated cover slips resulted in modest but similar reductions in the mean number
of foci in both exposed and unexposed cells and in reduced experimental
variability, so we utilized chambered slides for all subsequent experiments. Based on preliminary
flow cytometry data, we estimated that about 60% of cells were in G0/G1 and
thus we used adjusted DAPI intensity as a measure of DNA content to exclude the
40% of cells in G2/M or S-phase for all analyses (data not shown).Preliminary dose- and time-response patterns
based on control cell line GM12154 for the mean number ofγH2A.X foci following
gamma irradiation. Two independent experiments, each containing two replicates
(~200 cells each), were used for the analyses. (a) Linear dose response at 0.5
hour following gamma irradiation (R2  =  0.99). Error bars represent ±1 SEM for two
experiments. (b) Non-linear time response to 1.5 Gy gamma irradiation. The time
point, NT, represents preexposure cells. Error bars represent  ±1 SEM for two
experiments.
(a)(b)We compared groups
of cell lines representing each of the DNA repair gene polymorphisms to the
group of cell lines used as a control (Figure3). All groups of cell lines exhibited
similar time-course response patterns for the mean number of γH2A.X foci per cell before and following
exposure to 1.5 Gy of gamma irradiation (Figure 3(a)). The mean number of foci increased about fourfold from before
exposure to 1 hour postexposure in all of the groups of cell lines. Average
number of foci remained more than twofold elevated in all groups of cell lines 3 hours after
exposure, but all groups returned to near-baseline levels by 24 hours.Summaries of gene-(nsSNP-)specific
temporal response patterns following 1.5 Gy gamma irradiation for three different response measures. Time points are
NT/preexposure, 1 hour, 3 hours, and 24 hours following irradiation. Error bars
represent 95% confidence limits.
(a) Mean number of foci. (b) Mean area of foci. (c) Mean intensity of foci.
(a)(b)(c)Although all cell lines appear by inspection
to have similar induction and disappearance ofγH2A.X foci, we statistically tested whether any
of the 5 nsSNPs differed from controls using a mixed-model regression
analysis with the number of copies of the variant allele at each nsSNP as
predictors. This approach can
accommodate cell lines that may be heterozygous or homozygous at a locus or
that may have variants at more than one locus. We considered three response
variables constructed as ratios of the number of foci at different time points:
induction of foci at 1hour compared to preexposure (T1/T0), the repair of this
damage over a 3-hour time course (T3/T1), and the persistence of damage at 24 hours
compared to preexposure levels (T24/T0). All groups of cell lines had similar estimates for these ratios, and we
saw no evidence that any group of cell lines differed from controls (Table 3).Table 3
Comparison of the number of foci at various time points (T0, before irradiation; T1, T3, T24, respectively, 1 hour, 3 hours, 24 hours, following irradiation.)
GenensSNPRatios of geometric means†T1/T0T3/T1T24/T0PNKPrs37391864.650.671.25BRCA1rs17999504.510.651.09LIG4rs18053883.940.641.08WRNrs13460443.830.641.11ATMrs18000574.200.621.31CONTROLS4.100.641.16†None of the ratios for any cell line panel representing
SNPs was statistically
significantly different from the corresponding ratio for controls. (P>.05, t-tests comparing coefficients from a mixed model regression
fitted separately to the base-2 logarithm of each ratio).In
addition to measuring the number of foci within cells, we also measured the
size and intensity of foci, again concentrating on ratio responses analogous to
those mentioned earlier. By one hour after exposure, the mean size of
individual foci increased by more than 75% compared to preexposure levels in
all groups of cell lines and remained elevated and virtually unchanged at 3 hours
following exposure (Figure2(b)). Even
24 hours after exposure, when the average number of foci per cell had returned
to near preexposure levels, the average size of the foci remained significantly
larger than that of cells before exposure (P<.0001 for all
groups, P=.0003 for PNKP). One
hour following exposure, mean intensity of foci increased more than twofold
over preexposure levels, and it decreased only slightly from the 1 hour levels
at 3 hours (Figure 3(c)). Mean intensity
remained significantly elevated after 24 hours in all groups of cell lines
except those with ATM and PNKP polymorphisms, although even these
two had elevated intensities at 24 hours.


## 4. Discussion

Recent resequencing efforts have greatly expanded the catalog of SNPs available for
study. This catalog is increasingly being used in focused epidemiologic studies
of cancer susceptibility genes and in broader genome-wide association studies. DNA
repair genes are appealing candidates to study both because rare mutations in a number
of these genes have been linked to cancer risk and because genomic instability and mutation are important
features of the cancer process [1, 2]. The
most prominent a priori candidate SNPs for disease causation are those
that lead to nonsynonymous amino acid changes, in particular, the small subset that
are predicted to alter functional protein domain structure. Relatively few of
these SNPs have been evaluated in epidemiologic studies, in part because minor
allele frequencies are often less than 5% and thus require large sample sizes for
adequate statistical power. Because of
the difficulty in carrying out functional studies, even fewer have been
evaluated using in vitro assays.Using several in silico prediction tools to
evaluate nsSNPs in DNA repair genes, we selected 5 SNPs with “possibly” or
“probably damaging” amino acid substitutions in genes that are involved in DSBR.ATM, BRCA1, LIG4, and WRN have well
known associations with cancer or genetic diseases that predispose individuals
to cancer [2, 27–29].Specific missense
mutations inLIG4 have been
associated with
LIG4 syndrome which results in increased radiosensitivity [27].
LIG4 plays an essential role in the NHEJ pathway by rejoining ends of DNA at DSB
sites. The “possibly damaging” variant, T9I, has been associated with a reduced
risk for multiple myeloma [30] but has not otherwise been evaluated
for functional effects.DefectiveWRN results in Werner syndrome which is
characterized by an increased risk of cancer and other age-related disorders [28, 29]. WRN is a member of the RecQ family of DNA helicases that has both
3′ to 5′ helicase and exonuclease activities and may limit nucleotide removal during NHEJ [31]. Although the WRN C1367R polymorphism is predicted to
be “probably damaging,” one functional study of enzymatic activity found little
effect [32].PNKP phosphorylates 5′ hydroxyl termini and dephosphorylates 3′ phosphate termini [33]. Although generally characterized
as a base excision repair gene, it is also involved in phosphate replacement at
damaged termini during NHEJ [34]. PNKP interacts with XRCC4 and loss of this
interaction results in a slower rate of DNA repair and increased
radiosensitivity [35]. The PNKP Y196N variant lies within the PNK39 protein domain, which in
turn is thought to play a role in the repair of single strand breaks caused by
exogenous agents, although the functional consequences of this variant have not
been previously characterized.BRCA1 mutations increase an individual
risk for breast and ovarian cancer [27]. In response to a DSB, BRCA1 promotes HR and suppresses NHEJ [36]. The BRCA1 polymorphism, Q356R, is located within the site of
interaction with the Mre11/Rad50/Nbs1 (MRN) complex [36]. This complex is important in
sensing and repairing
DSBs [36], although two epidemiologic
studies of the BRCA1 Q356R
polymorphism failed to find an association with ovarian cancer risk [7, 37].Inactivation ofATM results in A-T which is associated
with increased radiosensitivity and risk for cancer [27]. ATM is activated in response to ionizing radiation and phosphorylates a number of
proteins involved in DSBR and checkpoint control, including p53, BRCA1, NBS1, CHK2, RAD9, MDM2, and H2AX [21, 38]. In addition, ATM is necessary for nucleosome disruption and histone loss at the
site of a DSB which may be necessary for proper recruitment of repair proteins [39]. To our knowledge, the only
study to date of the ATM variant P1054R
found no evidence for association with radiosensitivity in breast cancer
patients [40].The induction and
elimination ofγH2A.X foci following 1.5 Gy irradiation was
remarkably consistent among controls and the 5 cell line panels representing
different DNA repair gene polymorphisms. Compared to preexposure levels, all
cell line panels showed statistically significant increases in number,
intensity, and area of foci at both 1-hour and 3-hour time points. The number
of γH2A.X foci proved to be the most sensitive
index of exposure at both 1 and 3 hours, showing larger fold changes over preexposure
levels than either intensity or area of foci. In addition, the number of γH2A.X foci showed a large and significant
decrease between 1 and 3 hours whereas mean intensity of foci showed only small
changes between these two time points and mean area of foci remained virtually
unchanged. Whereas the number of foci had returned to baseline at 24 hours
following exposure, both intensity and area of foci remained significantly elevated
compared to preexposure levels for controls and most SNP panels.Our study is limited both by the number of
cell lines that constitute each panel and by the fact that in many cases
homozygotes were so rare that we could only study heterozygous individuals. We
cannot rule out the possibility that the nsSNPs that we evaluated might have
subtle effects on repair of DSBs beyond the sensitivity of our assay and sample
size. In addition,
it is possible that alternative DNA repair pathways could compensate for
decreased function and thus mask subtle functional effects, or that the nsSNPs we
evaluated could have other functional consequences,for example, in repair fidelity, that we have not assessed.Genome-wide association studies are becoming
increasingly popular, as is the use of endophenotypes to better understand the
etiology of complex diseases. However, difficulties in measuring these
intermediate phenotypes on a large scale can often limit selection. The
measurement ofγH2A.X foci formation is a useful tool for
evaluating the induction and repair of DSBs. Development of this assay for
large scale use could be made possible by using chamber slides with multiple
wells, microscopes with automated slide feeders and imaging capacities, and by
reducing the manual labor associated with cell culture. The ability to focus on
endophenotypes, such as DSBR, in genetic association studies may help us to
better understand the factors that predispose individuals to cancer.



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*Source: 387423-2009-03-12.xml*