GENE-38931; No.

of pages: 6; 4C:
Gene xxx (2013) xxxxxx

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Methods paper

Molecular defects identied by whole exome sequencing in a child with

Fanconi anemia
Zhaojing Zheng a, Juan Geng a, Ru-en Yao a, Caihua Li b, Daming Ying c, Yongnian Shen c, Lei Ying c,
Yongguo Yu c,, Qihua Fu a,
a
b
c

Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, PR China
Center for Genetic & Genomic Analysis, Genesky Biotechnologies Inc., Shanghai, PR China
Department of Internal Medicine, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, PR China

a r t i c l e

i n f o

Article history:
Accepted 9 August 2013
Available online xxxx
Keywords:
Fanconi anemia
FANCA
Whole exome sequencing
Molecular diagnostics

a b s t r a c t
Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations,
and an increased susceptibility to malignancy. At least 15 genes have been identied that are involved in the
pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to
characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing
was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation
was found (c.3971CNT, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA
gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identication
of disease-causing mutations in rare genetic disorders such as Fanconi anemia.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Fanconi anemia (FA) is a rare inherited syndrome characterized
by developmental defects, short stature, bone marrow failure, and an
increased risk to malignancies. Fifteen genetic subtypes of FA have
been identied so far (de Winter and Joenje, 2009; Kim et al., 2011;
Stoepker et al., 2011; Vaz et al., 2010). Approximately 85% of patients
are classied into subtypes A, C, or G, while the remaining 15% of
patients have one of the other 12 subtypes. All subtypes but FA-D1
and FA-N appear to t within the classical FA phenotype. Cells derived
from FA patients are hypersensitive to chromosomal breakage induced
by DNA inter strand cross-linking agents (ICLs) such as mitomycin C
(MMC) or diepoxybutane (DEB) (Rosendorff and Bernstein, 1988) and
assessment of this cellular hypersensitivity is the classic diagnostic
test for FA. However the chromosomal breakage test is positive in only
~10% of patients (Gille et al., 2012) and occasionally the test gives
false positive results in other genetic disorders, such as Nijmegen
Abbreviations: FA, Fanconi anemia; BRCA2/FANCD1, breast cancer 2/Fanconi anemia
group D1; PALB2/FANCN, partner and localizer of BRCA2/Fanconi anemia group N; ICLs,
interstrand cross-linking agents; MMC, mitomycin C; DEB, diepoxybutane; PGD, preimplantation genetic diagnosis; MLPA, Multiplex Ligation-Dependent Probe Amplication;
NGS, next generation sequencing; WES, whole exome sequencing; FANCA, Fanconi anemia
group A; CFU-GM, colony forming unit-granulocyte, monocyte; BFU-E, burst forming uniterythroid; CsA, cyclosporin A; SCGE, single cell gel electrophoresis; SNV, single nucleotide
variations; IGV, Integrative Genomics Viewer; HGMD, the Human Gene Mutation Database;
ESP, Exome Sequencing Project.
Corresponding author. Tel.: +86 21 38626161x5189; fax: +86 21 58756923.
Corresponding author. Tel.: +86 21 38625559; fax: +86 21 58756923.
E-mail addresses: yuyongguo@shsmu.edu.cn (Y. Yu), qfu@shsmu.edu.cn (Q. Fu).

breakage syndrome (NBS; MIM #251260) and Roberts syndrome

(RBS, MIM #268300).
Mutation analysis is available for all genes known to be associated
with FA, however this analysis is often complicated by many factors.
The quantity of genes to be analyzed, the large number of possible
mutations in each gene, the presence of large insertions or deletions
in some genes, as well as the large size of many of the FA-related
genes can make mutation analysis problematic. If the complementation
group has been established, the responsible mutation can be determined
by sequencing the corresponding gene. However, complementation
analysis is limited by the availability of cloned DNA from known FA
genotypes. Some centers recommend sequencing of all genes without
prior evaluation by complementation analysis since this analysis is only
performed in a limited number of research laboratories (Ameziane
et al., 2008). Sanger sequencing and multiplex ligation-dependent
probe amplication (MLPA) are commonly used for mutation testing in
FA patients (Gille et al., 2012), however they are rather laborious, time
consuming and expensive. Over the last few years, next generation sequencing (NGS) has revolutionized the approach by which we examine
the genetic causes of rare, single gene disorders (Bamshad et al., 2011).
Recently, NGS has become more common in the clinical diagnostic
arena and has been transforming the practice of molecular diagnostic
testing. Proof-of-principle studies have also been demonstrating the
feasibility of using whole exome sequencing (WES) for testing in a clinical setting (Calvo et al., 2012; Dixon-Salazar et al., 2012; Need et al.,
2012). WES will likely become more commonly used for clinical genetic
testing due to its lower sequencing cost and greater bioinformatic processing power. It may also improve the diagnostic yields for rare genetic

0378-1119/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.gene.2013.08.031

Please cite this article as: Zheng, Z., et al., Molecular defects identied by whole exome sequencing in a child with Fanconi anemia, Gene (2013),
http://dx.doi.org/10.1016/j.gene.2013.08.031

Z. Zheng et al. / Gene xxx (2013) xxxxxx

disorders. In the current study we detected the compound heterogeneous mutations in FANCA using whole exome sequencing in clinical
settings in an FA patient.

determine the purity and concentration of the gDNA in the sample.

The gDNA library was prepared using the TruSeq DNA Sample Preparation Kit (Illumina, USA) in accordance with the manufacturer's
instructions.

2. Materials and methods

2.1. Case report

2.3. Whole exome sequencing

A male patient, aged 4 years and 11 months, was born to a healthy

non-consanguineous parents. The patient had left hand preaxial
polydactyly and an irregular hypo-pigmentation spot on the left back
trunk. Surgery to correct the thumb deformity was performed when
the patient was 6 months of age. The patient's development was not
ofcially tested, but was estimated to be within normal range. The
patient's family history was unremarkable. The clinical features are
summarized in Table 1. In December 2010, the patient was incidentally
found to have thrombocytopenia. Biochemistry, ultrasonography, and
radiography did not reveal any signicant abnormalities. Bone marrow
aspirate examination showed normal cellularity with markedly decreased hematopoietic cells and normal morphology. Biopsy revealed
aplastic bone marrow with no signicant myelobrosis. No chromosomal aberration was detected by routine G-banding karyotype analysis
using peripheral lymphocytes. An in vitro assay for hematopoietic progenitor cells showed that the patient's colony forming unit-granulocyte,
monocyte (CFU-GM), burst forming unit-erythroid (BFU-E), CFU-E,
and CFU-mix were low and a diagnosis of aplastic anemia was made.
The patient was treated with cyclosporin A (CsA) and androgen for
10 months, however the patient did not respond well and the treatment
was discontinued. Traditional Chinese medicine was subsequently used
to treat the patient. In March 2012, a single cell gel electrophoresis
(SCGE) was done using peripheral lymphocytes; the patient was found
to have increased spontaneous and MMC-induced chromosomal breakage (Fig. 1) and a diagnosis of FA was made, however, the complementation group was not identied.
The Ethics Committee of the Shanghai Children's Medical Center
reviewed and approved this study. Written, informed consent was
obtained from the patient's parent.

In solution exome enrichment was done using the TruSeq Exome

Enrichment kit (Illumina, USA), according to the manufacturer's
instructions. The enriched DNA samples were sequenced via 2 100
paired-end sequencing using a Hiseq2000 Sequencing System (Illumina,
USA). Illumina Sequencing Control v2.8, Illumina Off-Line Basecaller
v1.8, and Illumina Consensus Assessment of Sequence and Variation
v1.8 software were used to produce 100 base pair (bp) sequence
reads.

2.4. Data analysis

Sequence reads were aligned to the human reference genome
(hg19) using BurrowsWheeler Aligner (Li and Durbin, 2010) with
default parameters. Variants were identied using Genome Analysis
Toolkit (GATK) (DePristo et al., 2011; McKenna et al., 2010) and VarScan
(Koboldt et al., 2009) software. Coverage analysis was determined using
the Picard software CalculateHsMetrics tool. Reads that matched exonic
regions, including exonintron-boundaries were analyzed. Single nucleotide polymorphisms (SNPs) and insertion/deletion (indels) analysis
was done using different ltering steps. Genes with at least two heterozygous changes in the DNA sequence were considered to be the most
likely to cause disease, though some homozygous variants were also
examined. Annovar (Wang et al., 2010), a software tool that accesses
and utilizes information from external databases to assess implications
and consequences of a given sequence alteration, was used to annotate
the resulting list of variants. Relevant mutations of all FA genes were
then prioritized manually.

2.2. Genomic DNA preparation

2.5. Filtering strategy

Genomic DNA was extracted from peripheral blood samples, collected

from the patient and his parent, using a Gentra Puregene Kit (Qiagen,
Germany). A spectrophotometer (NanoDrop, USA) was used to

The variant detection frequency was set at a minimum of 20% of the

reads covering any aberration. A minimum coverage by 10 reads was set
as the threshold for any variant to be considered a real mutation. In each
case, all variants listed in the most recent version of the NCBI (National
Center for Biotechnology Information) dbSNP database were excluded,
as well as silent mutations. Low frequency frameshift and truncating
mutations in any FA gene were considered to be pathogenic. Unreported
non-synonymous amino acid variants were analyzed by MutationTaster
(http://www. mutationtaster.org), Polyphen-2 (http://genetics.bwh.
harvard. edu/pph2) and SIFT (http://sift.jcvi.org) to assess any potentially damaging effects. Variants that passed these ltering steps were
considered to be the most likely to cause disease and were subsequently
validated by Sanger sequencing.

Table 1
Summary of clinical features in a patient with Fanconi anemia.
Clinical features

Status

Low birth weight

Microsomia
Skin

Absent
Absent
Generalized hyper-pigmentation
An irregular hypo-pigmentation spot
Preaxial polydactyly of left hand
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Thrombocytopenia preceded anemia
Pancytopenia-generally worsens over time
Severe bone marrow failure
112.2
20
50

Skeletal
Craniofacial
Eyes
Renal
Gonads
Developmental delay
Ears
Cardiopulmonary
Gastrointestinal
Central nervous system
Hematological

Current height(cm)
Current weight(Kg)
Head circumference(cm)

2.6. Validation by Sanger sequencing

Sanger sequencing was used to validate the candidate variants
identied above. PCR primers were designed with the Primer3
tool (http://frodo.wi.mit.edu/primer3/) so that they contained
the mutation sites and their anking regions (PCR primers, PCR
reaction conditions are available upon request). Sequencing was
achieved using ABI Prism 3730xl DNA Sequencer in combination
with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.1
(Applied Biosystems, USA). Sequences were assembled and analyzed
with Mutation Surveyor software (SoftGenetics, USA).

Please cite this article as: Zheng, Z., et al., Molecular defects identied by whole exome sequencing in a child with Fanconi anemia, Gene (2013),
http://dx.doi.org/10.1016/j.gene.2013.08.031

Z. Zheng et al. / Gene xxx (2013) xxxxxx

Fig. 1. Increased chromosomal instability was demonstrated in the patient with Fanconi anemia. a: Single cell gel electrophoresis assay (comet assay); b: Results of comet assay analyzed by
CASP and SPSS13.0; c: Mitomycin C (MMC)-induced chromosomal breakage in peripheral lymphocytes.

3. Results
3.1. NGS data
A total of 97,730,000 reads were obtained. The average target coverage was 66.27. Of these, 91.74% of reads had a Phred-like quality score
(Q score) of greater than 20 and 84.21% of reads had a Q score of greater
than 30. The proportion of target bases with read depths of 2, 10,
20, and 30 were 95.42%, 89.70%, 80.96%, and 70.78%, respectively.
There were 65,108 single nucleotide variations (SNVs) in the exome
prior to ltering, with 9429 SNVs occurring in coding regions.
3.2. Identication of disease-causing mutations
In total, WES revealed four heterogeneous mutations in all FA genes
in the patient (Table 2). Of note, two heterogeneous mutations were
detected in FANCA gene. They included the single-base substitution
c.3971CNT in exon 40, resulting in the amino acid change p.P1324L
(Fig. 2), and the 7-bp (GGGCTGT) deletion c.989_995del in exon 11,
leading to a premature stop codon at amino acid p.332 (p.H330LfsX2)

Table 2
Summary of variations in FA genes revealed by whole exome sequencing in the patient
with FA.
Gene

SNP ID

Chrs Position

FANCA rs182657062 16
FANCA
16
FANCL rs149731356
2
SLX4
rs36003440
16

89,805,925
89,862,325
58,392,880
3,633,395

Nucleotide

Protein

c.3971CNT
c.989_995del
c.685ANG; c.670ANG
c.4856CNT

p.P1324L
p.330_332del
p.T229A; p.T224A
p.P1619L

(Fig.3). Sanger sequencing conrmed the two mutations in gDNA

from the patient. We also conrmed the paternal segregation of
p.P1324L, whereas the frameshift mutation p.H330LfsX2 was present
in the maternal gDNA by Sanger sequencing.
The single-base substitution c.3971CNT mutation had been previously
reported and its pathogenicity well established (Adachi et al., 2002);
however, the deletion mutation c.989_995del had not yet been reported
in the Human Gene Mutation Database, the Fanconi Anemia Database,
the 1000 Genomes Database, or the NHLBI GO Exome Sequencing
Project (ESP) Database at the time this study was conducted. The transcript of novel c.989_995del mutation was predicted to be degraded by
the nonsense-mediated mRNA decay (NMD) pathway in silico analysis.
The pathogenic clue was further revealed by the increased genomic
instability of peripheral lymphocyte from the carrier of this novel
mutation in comet assay.
4. Discussion
Although the number of patients with FA is relatively low, the
number of different pathogenic variants described for the FANCA gene
is high since FA-A is usually associated with private FANCA mutations
in individual families (Bouchlaka et al., 2003; Demuth et al., 2000;
Levran et al., 2005; Savino et al., 2003; Wijker et al., 1991; Yagasaki
et al., 2004). The mutation types are heterogeneous and include point
mutations, small insertions/deletions, splicing mutations, and large
intragenic deletions. Molecular characterization and conrmation of the
complementation groups for an FA gene mutation in each FA case can
be time and labor intensive. Castella et al. (2011) proposed a strategy,
based on the Spanish mutational spectrum, designed to optimize FANCA
mutation screening. However, using this mutation screening strategy,

Please cite this article as: Zheng, Z., et al., Molecular defects identied by whole exome sequencing in a child with Fanconi anemia, Gene (2013),
http://dx.doi.org/10.1016/j.gene.2013.08.031

Z. Zheng et al. / Gene xxx (2013) xxxxxx

Fig. 2. Identication of a non-synonymous mutation in FANCA by exome sequencing. a: Visualization of FANCA sequencing reads containing P1324L mutation with the Integrative
Genomics Viewer (IGV); b: Conrmation of c.3971CNT mutation in the patient and his father by Sanger sequencing.

the c.3971CNT mutation in exon 40 and the novel c.989_995del mutation

in exon 11 described in this study would not have been identied until
the last round of mutation screening. Ameziane et al. (2012) successfully
identied the BRCA2, FANCD2, FANCI and FANCL mutations in unclassied
FA patients with a target sequencing approach. Knies et al. (2012) reported that, when carefully applied, WES could provide both complementation group conrmation and molecular characterization for an FA gene
mutation in a single tube approach and that this gives WES a competitive
advantage over classical genetic testing. Target sequencing provides
deeper coverage, requires less time, and is less costly when compared to
WES (Ameziane et al., 2012); however, the NGS panel for FA genes is
not yet commercially available. Data obtained by WES could be used for
the screening of FA patients in order to nd other candidate genes and
mutations since there are patients that still cannot be assigned to any of
the known complementation groups.
As expected, the quantity of variations detected by WES was very
high in the current study. Thus we utilized a prioritization approach
that followed a recessive inheritance mode, in order to increase the
productivity in the analysis of molecular diagnosis of FA.
According to the Fanconi Anemia Mutation Database and the
mutational spectrum in Spanish reported by Castella et al. (2011), the

majority of mutations found in FANCA are missense mutations and

small insertions/deletions that are distributed all along the gene. The
c.3971CNT mutation has been previously reported in the Fanconi
Anemia Mutation Database and its pathogenicity has been well
established (Adachi et al., 2002). The transcript of novel c.989_995del
mutation was predicted to be degraded by the nonsense-mediated
mRNA decay (NMD) pathway. Furthermore, the pathogenic clue of
the compound heterogeneous mutations was supported by the increased genomic instability to DNA ICLs observed in the patient. It's consistent with results from previous studies on other mutations (Adachi
et al., 2002; Castella et al., 2011; Garcia-Higuera et al., 2000). Therefore,
DNA damage signaling and other possible nuclear FANCA functions
may be completely abolished in all FANCA mutants, regardless of the
mutation type.
WES is an effective molecular diagnostic approach for FA in the
clinical setting. WES reduces turnaround time, the number of assays,
and the cost for reliable detection of disease-causing mutations. Since
the cost of enrichment and sequencing procedures continue to decrease, we expect that WES would replace Sanger sequencing and
MLPA as the rst tier test for patients with rare monogenic diseases
such as FA.

Please cite this article as: Zheng, Z., et al., Molecular defects identied by whole exome sequencing in a child with Fanconi anemia, Gene (2013),
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Z. Zheng et al. / Gene xxx (2013) xxxxxx

Fig. 3. Identication of a heterozygous deletion mutation in FANCA by exome sequencing. a: Visualization of FANCA sequencing reads containing the c.989_995del mutation with the
Integrative Genomics Viewer (IGV); and b: Conrmation of the frameshift mutation in the patient and his mother by Sanger sequencing.

Conict of interest
There's no conict of interest.
Acknowledgments
We thank the patient and his parent for consenting to participate
in this study. This study is supported by a grant from the Shanghai
Municipal Health Bureau New 100 Talents Program (No. XBR2011046,
to QHF), and a grant from the Shanghai Science & Technology
Commission Major Project (No. 11dz1950300, to YGY).
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