ARTICLE

ERCC6L2 Mutations Link a DistinctBone-Marrow-Failure Syndrometo DNA Repair and Mitochondrial Function

Hemanth Tummala,1,6 Michael Kirwan,1,6 Amanda J. Walne,1 Upal Hossain,1,5 Nicholas Jackson,2

Corinne Pondarre,3 Vincent Plagnol,4 Tom Vulliamy,1,7,* and Inderjeet Dokal1,5,7

Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are

first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations

affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells sig-

nificantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and

camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which

significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate

to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen

species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knock-

down cells and abolished ERCCGL2 traffic to themitochondria and nucleus in response to this DNA-damaging agent. Collectively, these

observations identify a distinct bone-marrow-failure syndrome due tomutations in ERCC6L2, a gene implicated in DNA repair andmito-

chondrial function.

Introduction

Bone-marrow-failure syndromes are a heterogeneous

group of life-threatening disorders characterized by the

inability of the bone marrow to make an adequate number

of mature blood cells.1,2 They can be variable in their

severity and can affect either one or all of the hemato-

poietic lineages. In some cases, individuals can be classified

into recognized syndromes, such as Fanconi anemia (FA

[MIM 227650])3 and dyskeratosis congenita (DC [MIM

305000]).4

In these diseases, the categorization of affected individ-

uals has been facilitated by the recognition of charac-

teristic features, such as the diagnostic mucocutaneous

symptoms of DC (abnormal skin pigmentation, nail dys-

trophy, and leukoplakia). Alternatively, specific genetic

and/or functional defects can be used in diagnosis, exem-

plified by the characteristically increased chromosomal

breakage observed in FA individuals. However, there are

also cases where the bone marrow failure is associated

with one or more extrahematopoietic abnormalities but

does not fit into a recognized syndrome and the underly-

ing genetic and functional basis is thus unknown.

The recent availability of next-generation sequencing

technology is making it possible to elucidate the genetic

basis and pathophysiology of uncharacterized human

diseases. From our repository of bone-marrow-failure cases,

we chose three genetically uncharacterized index cases in

whom to perform exome sequencing with the aim of iden-

1Blizard Institute, Barts and The London School of Medicine and Dentistry,

Haematology, University Hospital, Coventry CV2 2DX, UK; 3Institute of Ped4University College London Genetics Institute, London WC1E 6BT, UK; 5Bart6These authors contributed equally to this work7These authors contributed equally to this work and are co-senior authors

*Correspondence: t.vulliamy@qmul.ac.uk

http://dx.doi.org/10.1016/j.ajhg.2014.01.007. �2014 by The American Societ

246 The American Journal of Human Genetics 94, 246–256, February

tifying variants in a common gene. They had trilineage

(erythroid, myeloid, and megakaryocytic) bone marrow

failure and came from consanguineous families; all three

also had developmental delay characterized by learning

disability, and two out of the three cases also had micro-

cephaly (Table 1). We hypothesized that this approach

would enrich for homozygous disease-causing mutations.

Material and Methods

Exome SequencingPeripheral-blood samples were obtained with written consent

under the approval of our local research ethics committee

(London – City and East). DNA extracted from these samples

was submitted to the Beijing Genomics Institute for exome

sequencing. Ten micrograms of genomic DNA was supplied and,

after passing quality control, was subjected to Agilent SureSelect

library preparation and exome enrichment before being

sequenced on the Illumina GAII system. Sequencing data were

processed through the Illumina pipeline. Variants were called

with the Genome Analysis Toolkit (GATK) v.2.7.4. All samples

were called jointly with an additional data set of 1,005 locally

sequenced exomes with unrelated conditions (University College

London Exomes [UCL-ex] Consortium) after BAM file reduction

as implemented by GATK using default options. We used the

Illumina TruSeq target region (5 200 bp on each side) for variant

calling. We followed the GATK best practices and implemented

variant recalibration with separate models for SNPs and indels.

We excluded read depth from our recalibration model owing to

the large read-depth variability generated by the heterogeneous

Queen Mary University of London, London E1 2AT, UK; 2Department of

iatric Hematology and Oncology, Lyon I University, Lyon 69008, France;

s Health NHS Trust, London E1 1BB, UK

y of Human Genetics. All rights reserved.

6, 2014

Table 1. Features of Index Cases Who Underwent Exome Sequencing in Comparison to Those of FA, CS, and DC

Features

Index Cases in This Study

Case 1 (Family 1) Case 2 (Family 2) Case 3 FA CS DC

Gender male female female male and female male and female male and female

Age at presentation (years) 12 19 9 - - -

Ethnic origin French Pakistani Pakistani varied varied varied

First-cousin parents yes yes yes some some some

Trilineage bone marrow failure yesa yesb yesc yes no yes

Learning difficulties and/ordevelopmental delay

yes yes yes yes yes yes

Microcephaly yes yes no yes yes yes

Cutaneous photosensitivity no no no no some some

Cancer no no no yes no yes

Mucocutaneous features no no no some some yes

Other clinical features yesd yese yesf yesg yesh yesi

Chromosomal breakage in PBlymphocytes after treatment withDEB or MMC

normal normal normal increased normal normal

Telomere length normal short short short ? very short

Abbreviations are as follows: CS, Cockayne syndrome; DC, dyskeratosis congenita; DEB, diepoxybutane; FA, Fanconi anemia; MMC, mitomycin C; PB, peripheralblood; and ?, unknown.aPeripheral-blood analysis at presentation showed hemoglobin at 99 g/l, white cell count at 2.43 109/l, platelets at 93 109/l, and very hypocellular bone marrow(Figure 1E).bPeripheral-blood analysis at presentation showed hemoglobin at 93 g/l, white cell count at 5.3 3 109/l, platelets at 33 3 109/l, and hypocellular bone marrowwith features of dysplasia.cPeripheral-blood analysis at presentation showed hemoglobin at 102 g/l, white cell count at 2.33 109/l, platelets at 1283 109/l, and hypocellular bone marrow.d‘‘Abnormal facies’’ and ear abnormalities.eFloppy in infancy.fCleft palate and craniosynostosis.gVariable other features, including skin, gastrointestinal, and renal abnormalities.hVariable other features, including skin photosensitivity.iVariable other features, including immune deficiency and cerebellar hypoplasia.

capture kits used in the multiple studies that form UCL-ex.

Variants with PASS filter and the top-level recalibration tranche

(VQSRTrancheSNP99.00to99.90) were retained. We used a

variant-quality threshold of 30 and a genotype (i.e., sample-based)

Phred-scaled quality threshold of 20, with the exception of hetero-

zygous calls, for whichwe found the errormodel overly permissive

and for which we used a more stringent genotype Phred quality

threshold of 40. After variant calling, we used ANNOVAR to

perform annotation, which included allele frequencies obtained

from the NHLBI Exome Sequencing Project. We used the Ensembl

genes and transcripts to annotate the functional effect of exonic

variants. Splice variants were flagged within 8 bp of the exon-

intron junction. All variants identified were validated by Sanger

sequencing on a 3130xl Genetic Analyzer with a BigDye Termi-

nator v.3.1 Cycle Sequencing Kit (Applied Biosystems).

H&E Staining and ImmunohistochemistryAll sections were stained with hematoxylin and eosin (H&E)

and immunohistochemistry. We incubated glycol-methacrylate-

embedded sections in Tris-buffered saline (TBS) for 1 hr and

further subjected them to epitope unmasking by using a heat-

induced epitope-retrieval system with citrate buffer (Vector labs)

to avoid crosslinking of the proteins formed by formaldehyde

fixation without decalicification. We blocked sections with 5%

goat serum in PBS to completely avoid the background staining.

The Americ

Primary-antibody incubations diluted in blocking solution

(1:500) were carried out overnight at 4�C, subsequently washed,

and then incubated with secondary antibody Alexa Fluor 488

(Invitrogen) for 1 hr at room temperature. Slices were washed in

TBS containing 0.1% Tween 20, mounted with vectashield

mounting reagent (Vector Labs) on a cover glass, and subjected

to fluorescence imaging.

Cell Culture and Plasmid TransfectionHumanA549, human embryonic kidney 293, HeLa, and 293Tcells

were cultured in Dulbecco’s modified Eagle’s medium (DMEM)

with 10% (v/v) fetal bovine serum (FBS; HyClone), 100 IU/ml

penicillin, and 100 mg/ml streptomycin (Invitrogen). All cells

were maintained at 37�C in a humidified incubator with 5%

CO2. For ectopic expression studies, both 293T and A549 cells

were transfected with the appropriate plasmids by electroporation

with a Genepulser MX (Biorad) at 400V, 125 mF, and N ohms in

optiMEM (Invitrogen).

Immunocytochemistry, Imaging, and Colocalization

StudiesIn brief, after appropriate treatment, cells grown on coverslips

were washed in warm PBS and fixed in 4% paraformaldehyde.

After fixation, cells were permeabilized with 0.1% Triton X-100

an Journal of Human Genetics 94, 246–256, February 6, 2014 247

(TX100) in PBS, quenched in 50 mM NH4Cl, and blocked in 10%

goat serum and 1% BSA in PBS containing 0.05% TX100 for 1 hr.

Cells were incubated in the primary ERCC6L2 antibody (Abcam)

and the corresponding goat anti-rabbit secondary antibody conju-

gated to Alexa Fluor 488/568 (Invitrogen) in blocking solution for

1 hr separately. Cells were washed three times in PBS containing

0.05% TX100 between primary and secondary antibody incuba-

tions and mounted with vectashield containing DAPI (Vector

Labs). In cells expressing GFP-tagged wild-type (WT) and variant

forms of ERCC6L2, endoplasmic reticulum (ER) staining was

performed with mouse BiP antibody (Calbiochem). Autophagy

vacuoles were stained with LC3b antibody (Santa Cruz). For

lysosomes, A549 cells were stained with LysoTracker Red

DND-99 (Invitrogen) at a 100 nM/ml concentration; mito-

chondria were labeled with MitoTracker Orange CMTM Ros

(Invitrogen) at 37�C before fixation. Images were collected with

an LSM710 laser scanning confocal microscope (Olympus) under

relevant laser excitation, and the emitted signals were visualized

with ZEN software (Zeiss). For colocalization analysis, the back-

ground over noncellular regions was subtracted prior to determi-

nation of Pearson’s correlation coefficient (r2) in a minimum of

12 cells in each of three independent experiments. Image process-

ing was limited to contrast enhancement.

siRNA Transfection, Real-Time PCR, and

Immunoblotting DensitometryFor small interfering RNA (siRNA) studies, A549 cells were seeded

out in 6-well plates at 3.0 3 105 cells per well in antibiotic-free

DMEM containing 10% fetal calf serum (Invitrogen) and were

transfected the following day with Lipofectamine RNAiMAX

(Invitrogen). A pool of two siRNAs (SASI_Hs02_00305873 and

SASI_Hs01_00164316, Sigma-Aldrich) was used at a 30 nM final

concentration for ERCC6L2 knockdown, and a nontarget siRNA

(AllStars Negative Control siRNA, QIAGEN) was used as a negative

control. Mock-transfected cells were treated identically but

without any siRNA. Knockdown of ERCC6L2 expression was

confirmed by quantitative real-time PCR. RNA was extracted

with the RNeasy Mini Kit (QIAGEN), and cDNA was synthesized

with SuperScript II Reverse Transcriptase (Invitrogen). Quantita-

tive real-time PCR was performed with TaqMan probes

Hs02758991_g1 for ERCC6L2 and Hs00418541_m1 for GAPDH,

and reactions were run on an ABI 7500 thermal cycler (Applied

Biosystems). For immunoblotting, 30 mg of BSA-quantified protein

was loaded, transferred, and analyzed with a WesternBreeze

Chromogenic Kit (Invitrogen) and antibodies against ERCC6L2

(Abcam) and b-actin (Abcam), the latter of which was used as

a loading control. We performed semiquantitative analysis of

raw immunoblots by scanning the colorimetric blots at 600 dpi

resolution to TIFF format files, which we subjected to densitom-

etry analysis software (GelPro) to identify quantitative changes

in protein levels.

Cell Viability and ROS EvaluationAfter 48 hr exposure to ERCC6L2-siRNA-lipofection complexes

(SASI_Hs02_00305873 and SASI_Hs01_00164316, Sigma-Aldrich)

and nontarget siRNA (1027280, QIAGEN), medium was replaced

with DMEM containing 10% FBS supplemented with mitomycin

C (MMC), camptothecin (CPT), etoposide (ETP), and Irofulven

over a specified dose range in the presence or absence of N-acetyl

cysteine (NAC) (all purchased from Sigma-Aldrich). After further

48 hr incubation at 37�C, cells were washed in PBS, propidium

248 The American Journal of Human Genetics 94, 246–256, February

iodide was added to the cell suspension to a final concentration

of 5 mg/ml, and the cells were analyzed for staining on an LSRII

Flow Cytometer (Becton Dickinson). For the evaluation of basal

reactive oxygen species (ROS), cells were labeled with ROS marker

dihydroethidium (DHE, Invitrogen) at a 5 mM concentration, and

the fluorescence was measured by flow cytometry. Real-time

changes in ROS fluorescence upon Irofulven treatment were

measured at 37�C with standard 520 nm excitation and 610 nm

emission at 2 min intervals for a minimum of 60 min with the

use of a FLUROstar Optima plate reader (BMG Labtech). All read-

ings were normalized to the basal level (time ¼ 0). We calculated

statistical significance by comparing the linear regression of the

two curves with GraphPad Prism 5 software (GraphPad).

Subcellular FractionationSubcellular fractionation was performed according to a previously

described protocol5 with a slight modification. In brief, 1 3 106

DMSO-treated A549 cells and cells treated for 3 hr with MMC

(33 nM) or Irofulven (100 nM) were lysed in ice-cold HEPES

containing 5 mg/ml digitonin (Sigma Aldrich) and centrifuged at

3,000 rpm for extraction of cytoplasmic protein. Cell pellets

were resuspended in 1% Triton-X-114 in ice-cold PBS containing

a cocktail of protease and phosphatase inhibitors and centrifuged

at 14,000 rpm at 4�C for pelleting nuclei; the resulting superna-

tants consisting of membrane proteins were mixed in sample

buffer. The pellet-containing nuclei were briefly washed three

times in ice-cold radioimmunoprecipitation assay buffer contain-

ing a cocktail of protease and phosphatase inhibitors (Roche);

sonication followed twice for 10 s at 50% pulse. The final mixture

was shaken gently on ice for 15 min, and the protein supernatant

was obtained by centrifugation of lysates at 14,0003 g for 15 min.

Fractionated lysates were verified with antibodies against cyto-

plasmic GAPDH (Abcam), nuclear TATA binding protein (Abcam),

and the ER membrane protein BiP (Calbiochem) by immunoblot-

ting as described above.

Isolation of Mitochondrial Enriched LysatesExtracts enriched for mitochondrial proteins were obtained

according to a previously described protocol.6 In brief, after treat-

ment with MMC or Irofulven, cell pellets were resuspended in ten

packed cell volumes of buffer A (1 mM TrisHCl, pH 7.4, 0.13 M

NaCl, 5 mM KCl, and 7.5 mM MgCl2), pelleted, and resuspended

again in six packed cell volumes of homogenization buffer B

(10 mM Tris-HCl, pH 6.7, 10 mM KCl, 0.15 mM MgCl2, 1 mM

PMSF, and 1 mM DTT). Cells were homogenized on ice, and the

homogenate was transferred into a tube containing one packed

cell volume of 2 M sucrose solution and mixed gently. The cell

mixture was centrifuged at 1,2003 g for 5 min for pelleting unbro-

ken cells, nuclei, and large debris. The supernatant was transferred

to another tube and centrifuged at 7,000 3 g for 10 min for pellet-

ing mitochondria. The resulting supernatant was saved as soluble

cytoplasmic lysate 1. The mitochondrial pellet was resuspended in

three packed cell volumes of mitochondrial suspension buffer C

(10 mM TrisHCl, pH 6.7, 0.15 mM MgCl2, 0.25 M sucrose, 1 mM

PMSF, and 1 mM DTT) and centrifuged again at 9,500 3 g for

5 min for repelleting the mitochondria. The resulting supernatant

wasmixed with cytoplasmic lysate 1, obtained above. The pelleted

mitochondria were mixed with 13 sample buffer boiled at 95�Cfor 5 min and subjected to immunoblotting analysis. Mitochon-

drial fractionated lysates were verified with mitochondrial COXIV

(Abcam) by immunoblotting as described above.

6, 2014

Figure 1. Truncating Mutations inERCC6L2(A and B) Shown are two families in whichERCC6L2 mutations segregate as an auto-somal-recessive trait. A Sanger sequencingtrace and the genotype of each individualare given; inferred genotypes are in paren-theses.(B) For family 2, a plus sign (þ) indicatesthe WT allele and a minus sign (�) indi-cates the mutant allele. The normalsequencing trace to the left of family 2comes from an unrelated individual.(C) The identified ERCC6L2 alterationsleading to premature truncation are indi-cated on a diagram of the protein; func-tional domains are also annotated.(D and E) H&E staining of bone marrowtrephine biopsies from an unrelated con-trol sample and the index case (case 1)from family 1 reveals the degree of hypo-plasia in this individual.(F and G) Immunostaining on these bonemarrow trephine sections revealed thepresence of ERCC6L2 in a normal unre-lated control, but it was not detected inthe affected individual.(H and I) Positive control staining forGAPDH antigen was observed in both sec-tions. Figures are representative ofdifferent images taken from different fieldsof view. Magnification is 403. The scalebar represents 50 mm.

gH2AX AnalysisFor evaluating the DNA-damage response, cells were processed for

immunocytochemistry as described above with an antibody

specific to gH2AX, and the corresponding goat anti-rabbit

secondary antibody was conjugated to Alexa Fluor 488

(Invitrogen) in blocking solution for 1 hr separately. Images were

acquired with a Leica Epi fluorescence microscope with

MetaMorph (Molecular Devices) image capture software under

relevant filters. Captured images were processed for Image J

analysis for acquiring gH2AX quantification, and the quantified

florescence value obtained from each field of view was normalized

to the number of cells stained by DAPI.

StatisticsIn bar graphs, a Student’s t test was used for determining signifi-

cant differences between control and experimental groups.

The significant change in ROS (Figure 5A; n ¼ 3) and data are

plotted 5 SEM. In line graphs, for each experimental data set,

linear regression was conducted for determining the best-fit line

describing the data from each independent experiment. The over-

all significance of cytotoxicity (e.g., Figure 3A and B) or in ROS

accumulation rate (e.g., Figure 5B) was then determined with a

Student two-tailed t test of slopes of the regression lines from

each data set (n ¼ 3 independent experiments performed in octu-

plicate). Where indicated, statistical analysis was performed with

GraphPad Prism software, and a p value < 0.05 was considered

statistically significant.

The Americ

Results

Exome Sequencing Identifies TruncatingMutations in

ERCC6L2

Exome sequencing revealed that two (cases 1 and 2,

Table 1) of the three cases we studied with bone marrow

failure and developmental delay had homozygous trun-

cating variants in ERCC6L2 (alias RAD26L; RefSeq acces-

sion number NM_001010895.2). No other genes with

biallelic rare or somewhat rare variants were found to be

shared among the three cases. Specifically, the ERCC6L2

variants identified were c.1963C>T (p.Arg655*)

(Figure 1A) and c.1236_1239delAACA (p.Thr413Cysfs*2)

(Figure 1B). To our knowledge, the c.1236_1239del variant

has not been reported in any publicly available database,

but c.1963C>T is present in the NHLBI Exome Variant

Server at a frequency of 6 in 12,998 alleles, although not

in the homozygous state. No loss-of-function (LOF)

variant in ERCC6L2 was detected in an additional data

set of 1,005 locally sequenced exomes (UCL-ex Con-

sortium). Owing to this rarity of LOF variants (<1 in

2,000 alleles), the presence of biallelic LOF variants in

two out of three cases is extremely unlikely (chi-square,

p< 10�10). On that basis, we considered that the ERCC6L2

mutations were the likely cause of disease in these two

an Journal of Human Genetics 94, 246–256, February 6, 2014 249

Figure 2. Truncating Mutations Affect the Localization and Degradation of ERCC6L2(A) Confocal-microscopy images demonstrate predominant cytoplasmic and nuclear localization of WT GFP-ERCC6L2 in A549 cells.(B and C) Both truncating variants of ERCC6L2 formed aggregates.(E–L) Colocalization in yellow was observed for both of the altered ERCC6L2 proteins with BiP antibody, which binds to the ER proteinBiP (E and F), LC3b antibody, which localizes to autophagic vacuoles (H and I), and LysoTracker, which stains lysosomes (K and L). Thecolocalization observed was not an artifact of cross-channel noise or bleed from a compliment channel. The Pearson correlationcoefficients for colocalization revealed r2 > 0.5 when measured for the entire cell with ZEN software (Zeiss).(D, G, and J) No colocalization was observed betweenWT ERCC6L2 and any of these organelle markers (Pearson correlation coefficientsr2 < 0.08).(M–O) Neither the WT nor altered forms of ERCC6L2 showed any colocalization with ubiquitin, as evidenced by the green stainingpattern (Pearson correlation coefficient r2 < 0.03). All panels are representative of images taken from different fields of view in threeseparate experiments. Images display DAPI (blue), GFP-tagged ERCC6L2 (green), and organelle-labeling markers (red). The scale barrepresents 30 mm.

individuals. No obvious disease-causing variant was

identified in the third case.

Bone Marrow of an Affected Individual Lacks

ERCC6L2

ERCC6L2 is located on chromosome 9 (9q 22.32) and spans

14 exons. Expasy prosite analysis of ERCC6L2 revealed an

N-terminal DEAH ATP-helicase domain and a catalytic

helicase C-terminal domain (Figure 1C). Pfam analysis

indicated that it belongs to the Snf2 family of helicase-

like proteins, which are involved in transcription regula-

tion, DNA repair, DNA recombination, DNA translocation,

and chromatin unwinding.7 ERCC6L2 is 712 amino acids

in length and has a predicted molecular weight of

81 kDa. Immunoblotting on human cell lines revealed a

72 kDa band (Figure S1A, available online), and the speci-

ficity of the antibody was verified by two siRNAs targeting

the ERCC6L2 sequence at two different regions. Both

siRNAs decreased the expression of mRNA, as analyzed

48 hr posttransfection by real-time PCR (Figure S1B), and

reduced protein levels, as detected by immunoblotting,

confirming the efficiency of this antibody in detecting

ERCC6L2 (Figure S1C). Immunostudies on A549 cells

250 The American Journal of Human Genetics 94, 246–256, February

revealed the presence of ERCC6L2 in both cytoplasmic

and nuclear compartments (Figures S1D–S1H).

H&E staining revealed that compared to those of an un-

related control, bone marrow trephine sections obtained

from the individual homozygous for the c.1963C>T

mutation were hypocellular (Figures 1D and 1E). Immuno-

histochemistry on these sections showed no detectable

staining for ERCC6L2 in the sample from the affected

individual, but it did show clear positive cells in the unre-

lated control (Figures 1F and 1G); both sections stained

positive for GAPDH (Figures 1H and 1I). These results indi-

cate that truncating mutations might have an impact on

ERCC6L2 stability.

Mislocalization andDegradation of ERCC6L2 Variants

To investigate the impact of truncating mutations on

ERCC6L2 stability, we expressed both N-terminal GFP-

fused WT and mutant plasmid cDNA constructs in human

cell lines. In human A549 cells,WTGFP-ERCC6L2 revealed

a continuous diffuse pattern throughout the cytoplasm

and nucleus (Figure 2A). In contrast, both truncated forms

of GFP-ERCC6L2 showed marked aggregate-like structures

(Figures 2B and 2C). The distinct aggregate pattern of the

6, 2014

Figure 3. ERCC6L2 Plays a Role in theDNA-Damage-Response Pathway(A and B) Compared to nontarget-siRNA-transfected cells, ERCC6L2-knockdowncells showed reduced survival after 48 hrtreatment with MMC and Irofulven in adose-dependent manner. Error bars repre-sent the SEM obtained from three inde-pendent experiments (one-way ANOVAwith Tukey’s test).(C and D) Compared to nontarget-siRNA-transfected cells, ERCC6L2-knockdowncells revealed gH2AX foci at basal level.(E and F) gH2AX foci were higher in num-ber in ERCC6L2-knockdown cells thanin nontarget-siRNA-transfected cells aftertreatment with Irofulven. Panels are repre-sentative of images taken from differentfields of view displaying gH2AX (green)and DAPI (blue). The scale bar represents5 mm.(G) The bar graph represents the quanti-fied gH2AX fluorescence value obtainedfrom each individual field of view andnormalized to the number of cells stainedby DAPI (n > 50) at each individualtime point. Error bars represent the SEM(Mann-Whitney test), derived from dataobtained from five fields of view at eachindividual time point from two indepen-dent experiments.

ERCC6L2 variants was not cell-type specific given that it

was also seen in 293T cells (Figure S2).

Further characterization of these variant ERCC6L2 aggre-

gates inA549cells revealed that theywere localized to theER

(Figures 2D–2F), autophagic vacuoles (Figures 2G–2I), and

lysosomes (Figures 2J–2L), as observed by costaining with

specific cell organelle markers. However, no colocalization

was observed with ubiquitin (Figures 2M–2O). Consistent

with the lack of ERCC6L2 staining in the affected individ-

ual’s bone marrow, these studies demonstrate that both

truncating alterations in ERCC6L2 impair its normal locali-

zation and thus result in aggregation in the ER and likely

degradation through ER-associated autophagy.8,9

ERCCL2 Knockdown Reduces Cell Viability after

Genotoxic Stress

Until now, the function of ERCC6L2 has not been defined

experimentally, but it has been shown to have a ubiquitous

expression profile (Figure S3). Among the Snf2 family of

proteins, it shares the greatest peptide sequence homology

with the chromatin-remodelling and DNA-repair proteins

ALC1, CHD1, CHD4, RAD54, ATRX, and ERCC6 (Figures

S4A and S4B). Mutations in ATRX (MIM 300032) have

been linked to X-linked mental retardation most often

accompanied by alpha-thalassemia (ATRX syndrome

[MIM 301040]),10 and mutations in ERCC6 (MIM

609413) have been shown to cause Cockayne syndrome

(CS [MIM 133540]), characterized clinically by dwarfism,

microcephaly, cachexia, and neurodegeneration.11–13

Functional studies on RAD54, ALC1, CHD1, and CHD4

in eukaryotic cells revealed their role in facilitating DNA

The Americ

repair through chromatin remodelling.14 Loss of ATRX

has been shown to cause genomic instability and altered

DNA-damage response.15 ERCC6 has also been shown to

play an important role in a subpathway of nucleotide

excision repair (NER), known as transcription-coupled

NER (TC-NER), and also in mitochondrial function.16–21

Considering the homology of these genes and the overlap

of the clinical features seen in our individuals (specifically

learning difficulties and microcephaly), we speculate that

ERCC6L2 might also play a role in the DNA-damage

response.

To test this, we attempted to mimic the truncating

mutations by using siRNA to knock down ERCC6L2

expression in human A549 cells. After successful knock-

down (Figure S1B), we exposed the cells to the following

clastogens: (1) MMC, a compound that causes interstrand

crosslinks that can be repaired by the FA pathway or NER,22

(2) Irofulven, a compound that specifically induces DNA

adducts recognized by TC-NER, but not global genome

NER,23 and (3) CPT and (4) ETP, which induce double-

stranded DNA breaks via inhibition of topoisomerase I

and II, respectively. After 48 hr exposure, cell viability

was assessed by flow cytometry. Compared to mock-trans-

fected cells or cells transfected with nontarget siRNA, cells

transfected with ERCC6L2 siRNA showed significantly

reduced viability after exposure to MMC (unpaired

Student’s two-tailed t tests, p < 0.01, Figure 3A) and

Irofulven (unpaired Student’s two-tailed t tests, p <

0.001, Figure 3B) in a dose-dependent manner. With CPT

or ETP, no difference was observed in cell survival between

cells transfected with ERCC6L2 siRNA and cells transfected

an Journal of Human Genetics 94, 246–256, February 6, 2014 251

with nontarget siRNA (Figure S5). The consistent increase

in MMC and Irofulven sensitivity suggests a role for

ERCC6L2 in the repair of interstrand crosslinks and specif-

ically in TC-NER of DNA damage. We note that there is

evidence suggesting a role for TC-NER in the repair of

MMC-induced interstrand crosslinks.24 Despite the fact

that ERCC6L2-knockdown cells also showed increased

susceptibility to ETP and, to a lesser extent, CPT, a similar

pattern was also observed in cells treated with nontarget

siRNA. These results prevent us from concluding that

ERCC6L2 also plays a role in DNA-repair pathways acti-

vated upon topoisomerase inhibition.

Knockdown of ERCC6L2 Induces a DNA-Damage

Response

Snf2 protein complexes have been shown to be involved

in the recruitment of gH2AX, a phosphorylated form of

histone 2A, to sites of DNA damage.25 Given the increased

sensitivity of ERCC6L2-knockdown cells to MMC and

Irofulven, we wanted to investigate the role of ERCC6L2

with respect to this marker of DNA damage. Immunostain-

ing with a gH2AX-specific antibody revealed the presence

of discrete foci in ERCC6L2-knockdown cells in the

absence of any genotoxic stress (Figures 3C and 3D).

Compared to nontarget-siRNA-transfected cells, cells

treated with Irofulven for 3 hr at a low concentration

(100 nM) showed a further significant increase in the level

of gH2AX (Figures 3E and 3F). The kinetics of the increase

in gH2AX was more pronounced in ERCC6L2-knockdown

cells than in nontarget-siRNA-transfected cells over time,

suggesting an early DNA-damage response (Figure 3G).

Collectively, these results indicate that ERCC6L2 plays a

role in the DNA-damage-response pathway and that its

depletion sensitizes cells to genotoxic agents.

ERCC6L2 Traffics to the Mitochondria and Nucleus

after Genotoxic Stress

To further characterize the response of ERCC6L2 to

DNA-damaging agents, we performed immunocytochem-

istry on A549 cells. Compared to the normal diffuse

cytosolic and nuclear staining pattern seen in control

DMSO-treated cells (Figure S6A), a distinct perinuclear

accumulation of ERCC6L2 was seen in the cells treated

with both MMC and Irofulven (Figures S6B and S6C).

Immunoblotting on subcellular fractionated protein

lysates obtained from these cells revealed a dramatic

translocation of ERCC6L2 from cytosolic to membrane-

ous compartments and, to a lesser extent, toward the

nucleus after treatment with MMC or Irofulven for 3 hr

(Figures 4A and 4B).

ERCC6L2 has a distinct 13 amino acid N-terminal

sequence that harbors a putative mitochondrial targeting

sequence, as predicted by MitoProt software.26 This

prompted us to investigate extracts enriched with mito-

chondria obtained from A549 cells, and indeed these

showed an higher level of ERCC6L2 in mitochondria

than in the cytoplasm in cells treated with MMC and

252 The American Journal of Human Genetics 94, 246–256, February

Irofulven (Figures S6D and S6E). Immunocytochemistry

on cells labeled with MitoTracker and DAPI confirmed

the predominant localization of ERCC6L2 in the mito-

chondria (Figures 4C–4Q) and nucleus (Figures 4F, 4K,

and 4P) after MMC and Irofulven treatment. These results

are particularly intriguing in light of recent data indicating

that ERCC6 might act as a sensor of mtDNA damage19–21

and the central role that mitochondrial dysfunction plays

in the process of normal human aging.27

ERCC6L2 Knockdown Increases Intracellular ROS

Translocation of ERCC6L2 to themitochondria in response

to DNA-damaging agents led us to investigate its role in

mitochondrial function. We used ERCC6L2-knockdown

cells and nontarget-siRNA-transfected cells to quantify

intracellular ROS by using the ethidium-based probe DHE.

After successful knockdown, cells were stained with DHE

and subjected to flow cytometry. Compared to cells trans-

fected with nontarget siRNA, ERCC6L2-knockdown cells

showed a significant increase in intracellular ROS

(Figure 5A). To further investigate the changes in intracel-

lular ROS, we induced genotoxic stress with Irofulven

(100 nM) and monitored the change in intracellular ROS

over time. After treatmentwith Irofulven, ERCC6L2-knock-

down cells showed a significant increase (p < 0.0001,

comparing the linear regression of two curves) in the

change of intracellular ROS level over time in comparison

to nontarget transfected cells (Figure 5B). These results indi-

cate that depletion of ERCC6L2 results in increased ROS.

The Response of ERCC6L2 to Genotoxic Stress Is ROS

Dependent

To investigate a possible role for ROS in sensitizing

ERCC6L2-knockdown cells to genotoxic stress, we used

NAC, a well-known antioxidant that scavenges ROS.

ERCC6L2-knockdown cells and nontarget-siRNA-trans-

fected cells were treated with Irofulven at a cytotoxic

concentration (650 nM, refer to Figure 3B) in the presence

of NAC at two different concentrations (5 and 10 mM).

After 48 hr, flow cytometry analysis revealed that

compared to ERCC6L2-knockdown cells treated with

Irofulven alone, ERCC6L2-knockdown cells treated with

Irofulven and NAC had a significant (p < 0.0001) reduc-

tion of cell death in an NAC-dose-dependent manner

(Figure 5C). ERCC6L2-knockdown cells also stained nega-

tive for gH2AX upon treatment with Irofulven in the pres-

ence of NAC (data not shown). In addition, NAC inhibited

the Irofulven-induced translocation of ERCC6L2 to mito-

chondria (Figure 5D) and the nucleus (Figure S7). These

results suggest that ERCC6L2 traffic to both mitochondria

and nucleus is ROS dependent.

Discussion

In this study, we report on two homozygous truncating

ERCC6L2 mutations identified in individuals with

6, 2014

Figure 4. ERCC6L2 Traffics to the Mitochondria and Nucleus after Genotoxic Stress(A) Immunoblotting was performed on subcellular fractionated protein lysates after MMC and Irofulven treatment with ERCC6L2antibody. Abbreviations are as follows: Cy, cytosolic; N, nuclear; andM, membraneous. Subcellular fractionation was verified by GAPDHas a cytoplasmic marker and BiP as a membrane marker.(B) The increased membraneous and nuclear localization of ERCC6L2 is represented graphically by extrapolation of the values acquiredfrom densitometry analysis.(C–Q) Confocal images show ERCC6L2 localization to the mitochondria and nucleus after genotoxic stress. Nuclear localization ofERCC6L2 (white arrows) showed a correlation with DAPI staining after MMC (I and K) and Irofulven (N and P) treatment but to a lesserextent with DMSO (D and F). Colocalization of ERCC6L2 to the mitochondria is represented in yellow for MMC (J and L) and Irofulven(O and Q) treatment, but not DMSO (E and G) treatment. The colocalization observed was not an artifact of cross-channel noise or bleedfrom a compliment channel. For colocalization analysis, the Pearson correlation coefficients (r2) of the relative distribution of the twochannels in mitochondrial regions of the cells revealed r2 > 0.482 after MMC treatment, r2 > 0.739 after Irofulven treatment, and r2 <0.03 after DMSO treatment. Images display MitoTracker (red), ERCC6L2 (green), and DAPI (blue). Panels are representative of imagestaken from different fields of view in three separate experiments. The scale bar represents 30 mm.

neurological dysfunction (developmental delay andmicro-

cephaly) and bone marrow failure (cases 1 and 2, Table 1

and Figure 1). These mutations cause impaired subcellular

localization and reduced stability of ERCC6L2 (Figure 2).

We show that ERCC6L2 acts as a DNA-damage-response

protein by exacerbating gH2AX foci formation in response

to DNA-damaging agents (Figure 3). Our data also demon-

strate that ERCC6L2 traffics to the mitochondria and

nucleus in a ROS-dependent fashion in response to DNA

damage (Figures 4 and 5).

The Americ

Given the deficiency of ERCC6L2 in the bone marrow

of individuals harboring LOF variants in ERCC6L2, it is

reasonable to suggest that their DNA-damage response is

impaired. Because a DNA-damage response is required

during cell proliferation and tissue maintenance, its

persistent impairment would result in a slow accumulation

of DNA damage. In addition to showing the dual traffic

of ERCC6L2 from the cytosol to the mitochondria

and nucleus after genotoxic stress, we have shown a

parallel increase in gH2AX foci and intracellular ROS in

an Journal of Human Genetics 94, 246–256, February 6, 2014 253

Figure 5. The Relationship among ERCC6L2, ROS, and Genotoxic Stress(A) Compared to cells transfected with nontarget siRNA, A549 cells transfected with ERCC6L2 siRNA showed an increase in intracellularROS levels. Error bars represent the SEM of three independent experiments performed in triplicate (**p < 0.01).(B) Changes in ROS levels upon Irofulven stimulation were monitored over time. Error bars represent the SEM of eight readings in threeindependent experiments.(C) Analysis with fluorescence-activated cell sorting revealed reduced cytotoxicity of Irofulven in ERCC6L2-knockdown cells in the pres-ence of NAC in a concentration-dependent manner. Error bars represent the SEM from three independent experiments.(D) Immunocytochemistry on A549 cells treated with Irofulven (650 nM) in the presence of NAC (10 mM). Images display MitoTracker(red), ERCC6L2 (green), and DAPI (blue). Panels are representative of images taken from different fields of view in three separate exper-iments. The scale bar represents 30 mm.

ERCC6L2-knockdown cells. Increasing ROS levels leading

to alteration in cellular homeostasis is considered to be of

major pathological significance in neurodegenerative dis-

orders28 and bone marrow failure.29

There is also evidence that the increase in ROS levels

and incompetent DNA-damage repair contributes to the

pathogenesis of bone-marrow-failure syndromes.30 For

example, although FA is primarily characterized by chro-

mosome instability, developmental abnormalities, and

cancer susceptibility, it also shows reduced mitochondrial

function and increased ROS production.31 CS and the

functionally related xeroderma pigmentosum (XP [MIM

278700, 610651, 278720, 278730, 278740, 278760, and

278780]) are also caused by an inability to repair DNA

damage, particularly UV-induced DNA crosslinking32 and

increased ROS production.32,33 Cells from CS individuals

and CS mouse models have also shown increased ROS

activity and accumulation of damaged mitochondria as

a result of defective mitochondrial clearance by auto-

phagy.19 Recently, studies on DC lymphoblasts have

revealed elevated levels of ROS and a pronounced DNA-

damage response leading to a proliferative defect and

apoptosis.34,35 It is also known that increased ROS levels

impair the self-renewal capacity of hematopoietic progen-

itor cells in bone marrow.31 Taking all this together, we

propose that an increase in ROS and the accumulation of

254 The American Journal of Human Genetics 94, 246–256, February

DNA damage are the underlying causes of the disease

observed in our affected individuals.

Although some features overlap with previously

described syndromes, the individuals reported in this study

also have significant differences (Table 1). For instance, FA

is characterized by abnormal chromosomal breakage, but

analysis of peripheral-blood lymphocytes in our affected

individuals did not identify such defects. The increased

sensitivity to MMC in FA is also much more pronounced

than that observed in our ERCC6L2-knockdown assays

(Figure 3A) and the primary cells of these individuals (Table

1). Equally, bone marrow failure is not a reported feature of

CS, although neurological abnormalities are present

in most cases.36 XP, on the other hand, is primarily charac-

terized by hypersensitivity to sunlight and subsequent

development of carcinomas, and only some cases present

with neurologic disease.36 Interestingly, a recent study re-

ported a variant in ERCC4 (XPF) in a CS individual with

overlapping features of XP (severe photosensitivity and

abnormal skin pigmentation) and FA (abnormal chromo-

somal breakage).37 Hoyeraal Hreidarsson syndrome (HHS

[MIM 300240]) is characterized by neurological dysfunc-

tion, microcephaly, and bonemarrow failure and is consid-

ered to be a severe form of DC,38 a telomere maintenance

disorder,39,40 which itself commonlymanifests withmuco-

cutaneous abnormalities and bone marrow failure.4 Our

6, 2014

individuals lacked the mucocutaneous features, immuno-

deficiency, and cerebellar hypoplasia typically seen in DC

and HHS and, in comparison to age-matched controls,

had variable telomere lengths when measured by multi-

plex PCR41 (Table 1 and Figure S8). It is also notable that

exome sequencing did not identify variations in any of

the known genes associated with bone marrow failure in

these individuals.

Because there is no precise match between all the fea-

tures in the cases reported here and other genetically

characterized disorders, we propose the presently described

disorder as a distinct bone-marrow-failure syndrome. We

also believe that our study highlights the combined roles

of intracellular ROS and DNA damage in driving bone

marrow failure.

Supplemental Data

Supplemental Data include eight figures and can be found with

this article online at http://www.cell.com/AJHG.

Acknowledgments

We would like to thank the families and clinicians who contrib-

uted to this research, as well as Martine French for providing

bone marrow slides. This work was funded by TheWellcome Trust

and Medical Research Council. We would also like to thank Gary

Warnes and Ann Wheeler for support and technical assistance in

fluorescence-activated cell sorting and imaging.

Received: November 13, 2013

Accepted: January 10, 2014

Published: February 6, 2014

Web Resources

The URLs for data presented herein are as follows:

1000 Genomes, http://www.1000genomes.org

ANNOVAR, www.openbioinformatics.org/annovar

BioGPS, http://www.biogps.org

dbSNP, http://www.ncbi.nlm.nih.gov/SNP/

GATK, http://www.broadinstitute.org/gatk/

MitoProt, http://ihg.gsf.de/ihg/mitoprot.html

NHLBI Exome Sequencing Project (ESP) Exome Variant Server,

http://evs.gs.washington.edu/EVS/

Online Mendelian Inheritance in Man (OMIM), http://www.

omim.org

Pfam, http://pfam.sanger.ac.uk

Prosite, http://www.expasy.ch/Prosite

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