Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice

Mutation Research 810 (2016) 30–39

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice Kenichi Masumura a,∗ , Naomi Toyoda-Hokaiwado a , Akiko Ukai a , Yoichi Gondo b , Masamitsu Honma a , Takehiko Nohmi c a

Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan c Biological Safety Research Center, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan b

a r t i c l e

i n f o

Article history: Received 9 August 2016 Received in revised form 20 September 2016 Accepted 27 September 2016 Available online 28 September 2016 Keywords: Germline mutation Whole-exome sequencing Inherited mutation Mutation frequency gpt delta mouse Transgenic rodent gene mutation assay

a b s t r a c t Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85 mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30 mg/kg) were added, and dose-response of inherited germline mutations was analyzed. Male gpt delta transgenic mice treated with ENU in three dose groups were mated with untreated females 10 weeks after the last treatment, and offspring were obtained. The ENU-treated male mice showed dose-dependent increases in gpt mutant frequencies in their sperm, testis, and liver. gDNA of one family (parents and four offspring) from each dose group was used for whole-exome sequencing, and unique de novo mutations in the offspring were detected. Frequencies of inherited mutations increased with dosage more than 25-fold in the highest dose group. The mutation spectrum of the inherited mutations showed characteristics of ENU-induced mutations, such as A:T base substitutions. No confirmed mutations were observed in the control group. Filtering using the alternate reads ratio resulted in the mutation frequencies and spectra similar to those obtained by the Sanger sequencing confirmation. These results suggest that direct sequencing analysis may be a useful tool to investigate inherited germline mutations induced by environmental mutagens. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Gene mutations induced in germline cells are heritable and thus may cause health disadvantages in future generations. The germline mutation rate in humans has been estimated at approximately 1 × 10−8 per base per generation [1–5]. Most inherited mutations are harmful or neutral at best [4]. Next-generation sequencing (NGS) technology has been widely used in medical genomics and many genes associated with disease have been identified by searching a single nucleotide mutation or structural variation in human genome [6]. De novo mutations contribute to a wide range of human disorders [7–10]. De novo germline muta-

∗ Corresponding author. E-mail address: [email protected] (K. Masumura).

tions and their health effects in future generations are important in the field of genetic toxicology and for regulatory assessment [11]. However, the availability of mutagenicity tests used in germline cells, especially for detecting inherited mutations, is very limited. For example, mouse-specific locus tests and mouse heritable translocation tests have been used to detect inherited mutations [12,13]. However, these tests use considerably large numbers (hundreds to thousands) of animals and are difficult to carry out. As an alternative, some genotoxicity and mutagenicity tests can be applied to germline cells, such as micronucleus assay, comet assay and expanded simple tandem repeat assay [14–16]. Transgenic rodent (TGR) gene mutation assays are useful for detecting mutations in both somatic and male germline cells [17]. We previously reported heritable mutations in the offspring of N-ethylN-nitrosourea (ENU)-treated mice using whole-exome sequencing and also developed a method to estimate the inherited germline

http://dx.doi.org/10.1016/j.mrgentox.2016.09.009 1383-5718/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).

K. Masumura et al. / Mutation Research 810 (2016) 30–39

mutation frequency [18]. ENU is an alkylating agent and is known as a germ cell mutagen that is associated with the highest frequency of mutations in pre-meiotic spermatogonial stem cells [19–21]. The progression time of the developing germ cells from spermatogonial stem cells to mature sperm reaching cauda epididymis is ∼49 days in mice. In our study, male gpt delta mice were treated with ENU and mated with untreated female mice 10 weeks after the last treatment. Therefore, the origin of the male-contributed haplotype of the offspring was germ cells exposed at the spermatogonial stem cell stage. In the previous study, genomic DNA (gDNA) was extracted from six mice, including the father, mother, and four offspring from each family of the ENU-treated and untreated mice. In total, 12 DNA samples were subjected to whole-exome sequencing followed by Sanger sequencing confirmation. The frequency of inherited mutations in the offspring of the ENU-treated family was significantly higher than that of the control family. These results suggest that ENU-induced de novo inherited mutations can be detected by NGS using small numbers of animals; however, only one family originating from one ENU-treated mouse was analyzed in the previous study [18]. In this study, we analyzed three ENU doses (10, 30, and 85 mg/kg, i.p., once per week for 2 weeks) to investigate dose-response of somatic and germ cell mutations as well as inherited germline mutations. In addition, the method of detection using whole-exome sequencing was improved and the robustness of the analyses was examined using different tools for mapping and variant calling. 2. Materials and methods 2.1. Animal manipulation and breeding Male and female gpt delta mice (C57BL/6J background) [22–24] were obtained from a breeding colony maintained at the National Institute of Health Sciences. The animal treatment employed in this study was approved by the Animal Care and Utilization Committee of the institute. Animal experiments 1 and 2 were performed as described below (Supplementary Fig. S1). Experiment 1 was described in a previously published study [18]. Briefly, nine-weekold male mice were treated with ENU (85 mg/kg body weight intraperitoneally, weekly on two occasions). To check for a period of infertility induced by ENU, male mice were mated with untreated female mice 6–7 weeks after the last treatment. Ten weeks after the last treatment, these same male mice were mated with different untreated females. Control male mice were treated with phosphate/citrate buffer as vehicle and mated with untreated females 10 weeks after the last treatment without a pre-mating. Five male mice were used in each group. After the collection of offspring, the male mice were sacrificed (28–30 week-old), and their tissues were collected and stored at −80 ◦ C. The mated females and offspring were also sacrificed at 28–33 weeks and 5 weeks, respectively. Experiment 2 was performed using the same protocol as experiment 1, but two different doses of ENU (30 and 10 mg/kg body weight) were used. The ENU-treated and vehicle-treated male mice were mated with untreated females 10 weeks after the last treatment. Five male mice were used in each group. Tissue samples were collected from each family and stored. 2.2. Reporter gene mutation assay gDNA was extracted from liver and testis using the RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA). Sperm DNA was extracted as previously described [18]. In brief, the cauda epididymis was sliced in phosphate-buffered saline (pH 7.4), filtered, and pelleted by centrifugation. The pellet was re-suspended in 1 × saline sodium citrate (SSC) and 0.15% sodium dodecyl sul-

31

fate (SDS). The lysate was centrifuged and the sperm pellet was suspended in 0.2 × SSC, 1% SDS, 1 M 2-mercaptoethanol, and 10 mM EDTA (pH 8.0), and then digested overnight with 0.5 mg/mL proteinase K at 37 ◦ C. DNA was isolated by phenol/chloroform extraction, ethanol precipitation, and re-suspension in TE buffer (pH 8.0). Lambda EG10 transgenes were rescued from gDNA by in vitro packaging reactions using Transpack Packaging Extract (Agilent Technologies). The gpt mutation assay was performed as described previously [25]. In brief, the rescued phages infect Escherichia coli strain YG6020, which express Cre recombinase to convert the transgene into a plasmid. The infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol (Cm) and 6-thioguanine (6TG). The plates were incubated for 4 days at 37 ◦ C to select colonies that harbored the plasmid carrying the mutated gpt gene. To determine the number of rescued plasmids, infected cells were also poured onto plates containing Cm without 6TG. The gpt mutant frequencies were calculated by dividing the number of 6TG-resistant colonies by the number of rescued plasmids. 2.3. Whole-exome sequencing analysis The exomes of each of the ENU-treated families and one control family were sequenced (Fig. 1). Each family comprised six mice, i.e., parents (male and female) and offspring (two males and two females). The 85 mg/kg ENU-treated and control families were randomly chosen from experiment 1 as described in [18]. The 10 and 30 mg/kg ENU-treated families were chosen from experiment 2. gDNA was extracted from the liver using a DNA Extractor WB Kit (Wako, Osaka, Japan). Liver DNA samples from 24 mice were subjected to whole-exome sequencing and data analyses was performed by Beckman Coulter Genomics (MA, USA) and Genaris Omics Inc. (Kanagawa, Japan). gDNA was fragmented using the Covaris DNA shearing system (Covaris, Inc., MA, USA). The entire mouse exome (49.6 Mb) was captured using a SureSelect Mouse All Exon Kit (Agilent Technologies) and sequenced by a Hiseq2000 (Illumina, CA, USA) with 100-bp paired-ends. Sequenced reads were obtained for 85 mg/kg ENU-treated and control families in the previous study [18] and for 10 and 30 mg/kg ENU-treated families in this study. Data analyses using 24 mice were newly conducted. The sequenced reads were mapped onto the reference sequence using CASAVA ELAND tool v.1.8.2 (Illumina) or Burrows-Wheeler Aligner (BWA) v.0.7.11 [26]. The reference sequence was C57BL/6J mouse genome: NCBI Build 37 (mm9) because the SureSelect Mouse All Exon Kit was designed using the same reference sequence. Mapped reads were rearranged using the Genome Analysis Toolkit (GATK) [27] and duplicated reads were checked by the Picard tool. For each animal, single nucleotide variants (SNVs) were called based on comparisons with the reference sequence using SAMtools v.0.1.18 [28] or GATK. The detected SNVs were annotated by SnpEff [29]. 2.4. Detection of de novo mutations and calculation of the frequency of inherited mutations in the offspring of ENU-treated and control males De novo mutations in the exomes of offspring were identified by Trio analysis, i.e., the SNVs were compared between the parents and offspring, and de novo mutations were identified as follows: (1) SNVs that were potentially transmitted from the parents to offspring were excluded. (2) SNVs found in only a single offspring (absent in the 23 other mice) were considered unique mutations and selected as candidate de novo mutations. (3) Only NGS genotype quality (GQ) scores greater than 20 (=99% accuracy) for a Trio, i.e., father, mother and offspring, were included. (4) The read depth, i.e., the number of sequenced reads that covered one nucleotide position, had to exceed a cut-off value in a Trio. The cut-off value was

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K. Masumura et al. / Mutation Research 810 (2016) 30–39

Vehicle 㽢2

ENU 10 mg/kg㽢2

ENU 30 mg/kg㽢2

ENU 85 mg/kg㽢2

䗢 䗠

䗢 䗠

䗢 䗠

43

29

44

50

52

䗢 䗠 8

62

99

䗢 䗢 䗠 䗠

䗢 䗢 䗠 䗠

䗢 䗢 䗠 䗠

䗢 䗢 䗠 䗠

501

523

606

991

502

503

504

524

528

529

607

610

611

992

994

995

Fig. 1. Mouse families used for whole-exome sequencing analyses. Each family had six mice comprised of the parents (one male and one female) and four offspring (two males and two females). Each animal ID is presented. gDNA was extracted from liver.

3. Results 3.1. Mutant frequencies in somatic and germline cells in ENU-treated mice Mutant frequencies in the liver, testis and sperm of the ENUtreated male mice were estimated by the gpt mutation assay (Supplementary Tables S1–S3). The mutant frequencies in the liver and sperm of 85 mg/kg ENU-treated mice and 5 control mice in experiment 1 were reported in a previous study [18]. Because there was no significant difference in the mutant frequency of the control mice between experiments 1 and 2, irrespective of tissue type, these data were merged and are presented in Fig. 2. The gpt mutant frequencies in the liver, testis and sperm of the three ENU doses were significantly higher than the control and increased with dose dependency. In the liver, the mutant frequency increased to 88.4 ± 25.4 × 10−6 in 85 mg/kg ENU-treated mice which was 37-fold higher than the control (2.0 ± 1.2 × 10−6 ). The mutant frequency in testis increased to 96.2 ± 27.8 × 10−6 which was 55-fold higher than the control (1.7 ± 1.9 × 10−6 ). The mutant frequency in sperm, increased to 44.4 ± 25.9 × 10−6 which was 18-fold higher than the control (2.4 ± 1.4 × 10−6 ). The mutant frequency in the liver of 30 mg/kg ENU-treated mice was significantly higher than the frequencies in testis and sperm (p < 0.05, Steel test). Significant

140

** 120

gpt Mutant frequency (x 10-6)

established as described in [18]. In brief, the ratio of the total number of mutation candidates in the ENU-treated group relative to that in the control group was plotted against the minimum read depth in a Trio. The read depth where the ratio peaked is the most sensitive setting for detecting a maximum fold-increase in the number of ENU-induced de novo germline mutations under the experimental conditions, and that read depth was selected as the cut-off value. (5) Only mutation candidates where more than 20% of total reads were alternate reads were included because lower alternate read ratios are not considered inherited heterozygous mutations. The alternate read ratio was defined as a ratio of the number of variant reads out of the total reads at a sequenced position. (6) Confirmation of the mutation candidates by Sanger sequencing was performed by TaKaRa BIO Inc. (Shiga, Japan) using an ABI3730 sequencer (Applied Biosystems by Life Technologies) with customdesigned PCR primers for each mutated position in the genomes of the offspring. (7) If Sanger sequencing confirmation was not performed, we added two selection filters instead (see Results). First, the ratio of alternate reads had to be more than 0.3 and less than 0.7, and second, the consensus sequence had to be homozygous and identical in the other 23 mice. The frequency of inherited germline mutations was calculated as follows. The number of de novo mutations was divided by the number of bases in the exome sorted with the same cut-off value, i.e., a minimum read depth and GQ score in a Trio.

**

100 80

** **

60

**

liver testis sperm

**

40

* * *

20

** **

0

0 mg/kg x2 (n=10)

10 mg/kg x2 (n=5)

30 mg/kg x2 (n=5)

85 mg/kg x2 (n=5)

Fig. 2. The gpt mutant frequencies in liver, testis and sperm of the ENU-treated male mice. The mutant frequencies are presented as average with standard deviations. Asterisks indicate significant differences versus the control group. Significant differences between tissues were observed in the 30 mg/kg ENU-treated group (*: p < 0.05, **: p < 0.01, Steel test). The mutant frequencies in the liver and sperm of 85 mg/kg ENU-treated mice and 5 control mice were obtained in a previous study [18].

differences between the tissues were not observed in the other ENU doses and controls. 3.2. Whole-exome sequencing and frequency estimates of de novo mutations (Analysis 1) One of the ENU-treated mice from each dose group (ID 8 for 85 mg/kg, ID 044 for 30 mg/kg and ID 029 for 10 mg/kg) and one control mouse (ID 43) were selected randomly. The gpt mutant frequencies in the livers of these mice were 74.1 × 10−6 , 23.4 × 10−6 , 4.8 × 10−6 and 1.2 × 10−6 , respectively. Entire families (both parents plus offspring) were used for whole-exome sequencing (Fig. 1). gDNA was prepared from the livers of 24 mice (4 families). Initial NGS read data comprising 9–24 gigabases (Gb) were obtained for each animal. The sequenced reads were mapped by ELAND onto the reference mouse genome sequence, where 35%–62% of the mapped reads were mapped onto the exon region. When 5 Gb of the sequenced data were mapped onto the 49.6-Mb exon region, it resulted in approximately 100-fold redundancy. The average read depth on the target was 112 (61–153 per sample). The proportion of target bases that had a depth of 10-fold or more was 85–97%. For each animal, SNVs and small indels were detected based on comparisons with the reference sequence by SAMtools. The SNVs were compared in the parents and offspring, and unique de novo mutation candidates in the offspring were scored. The ratio of the total number of mutation candidates in the ENU-treated groups relative to that in the control group was plotted against the minimum read depth in a Trio (Fig. 3). The ratio increased with the read depth

K. Masumura et al. / Mutation Research 810 (2016) 30–39

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Table 1 Frequencies of inherited mutations in the offspring of ENU-treated fathers in analysis 1 (ELAND + SAMtools). Average ± SD

G1 ID

P ID

M ID

No. of bases sorted in exome (depth ≥ 53)

NGS-called mutation (depth ≥ 53)

Confirmed mutation (by Sanger seq.)

Mutation frequency (×10−8 /base)

ENU 85 mg/kg × 2 male 991 male 992 female 994 female 995

8 8 8 8

99 99 99 99 total

12,138,578 8,215,968 12,135,801 12,157,990 44,648,337

14 14 18 14 60

12 11 18 14 55

98.9 133.9 148.3 115.2 123.2

124.1 ± 21.6*

62 62 62 62 total

18,051,273 18,142,166 18,195,560 18,234,173 72,623,172

8 4 8 3 23

8 4 6 3 21

44.3 22.0 33.0 16.5 28.9

28.9 ± 12.3*

52 52 52 52 total

7,151,170 7,069,851 7,016,051 7,157,517 28,394,589

1 0 2 0 3

0 0 2 0 2

0.0 0.0 28.5 0.0 7.0

7.1 ± 14.3

50 50 50 50 total

5,527,345 5,179,852 5,322,222 5,169,792 21,199,211

0 1 0 0 1

0 0 0 0 0

0.0 0.0 0.0 0.0 0.0

<4.8

30 mg/kg × 2 606 male male 607 female 610 female 611

44 44 44 44

10 mg/kg × 2 523 male 524 male 528 female 529 female

29 29 29 29

Vehicle × 2 male 501 male 502 503 female female 504 *

43 43 43 43

Asterisks indicate significant differences versus control (p < 0.05, Steel test).

Assumed calculation*

70 60

Fold increase

10mg / 0mg 50

30mg / 0mg

40

85mg / 0mg

30 20 10 0 0

10

20

30

40

50

60

prised 5.2–18.2 Mb per offspring. In the ENU and control groups, 87 candidate mutations were detected from 16 offspring (Table 1). Those candidate mutations in the offspring genomes were examined by Sanger sequencing and 90% (78/87) were confirmed as true mutations. All mutations were identified as heterozygous point mutations (Supplementary Table S4). The frequencies of inherited mutations in the 85, 30, and 10 mg/kg ENU-treated groups were 124.1 × 10−8 , 28.9 × 10−8 , and 7.1 × 10−8 per base, respectively. Because no confirmed mutations were detected in the control offspring, the mutation frequency (MF) in the control group was likely less than 4.8 × 10−8 per base. The MFs increased with dose dependency. In the higher two dose ENU-treated groups, the MFs were significantly higher than that of the control group (p < 0.05, Steel test).

70

Read depth ≥ XX

Fig. 3. Fold increase of the number of mutation candidates detected in the offspring of ENU-treated and control fathers in analysis 1 (ELAND + SAMtools). Ratio of the total number of de novo mutation candidates in four offspring from the ENU and control groups were plotted against minimum read depth in a Trio (father, mother and each offspring). The arrow indicates the peak of the fold-increase of the mutation candidates. When read depth was ≥53, the fold-increase was 60, 23 and 3 for 85, 30 and 10 mg/kg ENU-treated groups, respectively. *When the minimum read depth was ≥55, no mutation candidate was detected in the 0 mg/kg control group. Therefore, fold increase was virtually calculated based on the assumption that the control group has one mutation.

and peaked when the read depth ≥ 53. The fold-increase at the peak was 60, 23, and 3 for 85, 30, and 10 mg/kg ENU-treated groups, respectively. This was the most sensitive setting for detecting ENUinduced de novo germline mutations under these experimental conditions. Therefore, a read depth ≥ 53 in a Trio was selected as the cut-off value to calculate the frequency of inherited mutations. The number of bases in the exome was calculated in each offspring using the same cut-off conditions (Supplementary Fig. S2). The nucleotide sequences that passed the cut-off conditions com-

3.3. Improved de novo mutation estimate without Sanger confirmation (Analysis 2) To improve the detection methods for inherited de novo mutations, NGS-called mutation candidates were further analyzed. In 87 mutation candidates detected by analysis 1, the ratio of number of alternate reads per total reads was scored (Fig. 4). The alternate read ratio peaked at around 0.5 and a minor peak was observed at less than 0.3. Interestingly, the majority of false positives that failed to be confirmed by Sanger sequencing had ratios less than 0.3. When inherited de novo mutations were heterozygous, the alternate read ratio were normally distributed at peak = 0.5. This means that possible false positives could be efficiently excluded by cutting both sides of the alternate read ratio. For example, by sorting as 0.3 ≤ ratio ≤ 0.7, 78% (7/9) of the false candidates were excluded and 97% (76/78) of the confirmed true mutations remained. This simple sorting method efficiently replaced confirmation by Sanger sequencing. To investigate the reproducibility and robustness of the detection methods for inherited de novo mutations, we re-analyzed the data using different software for mapping (BWA) and SNV calling

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K. Masumura et al. / Mutation Research 810 (2016) 30–39

0.3

0.7

25 20 15

Confirmed mutation

10

False positive 5

≤ 1.0

≤ 0.9

≤ 0.95

≤ 0.8

≤ 0.85

≤ 0.7

≤ 0.75

≤ 0.6

≤ 0.65

≤ 0.5

≤ 0.55

≤ 0.4

≤ 0.45

≤ 0.3

≤ 0.35

0

0.2 ≤ 0.25

No. of mutation candidates

30

Alternate read ratio Fig. 4. Distribution of the mutation candidates with the alternate read ratios in analysis 1 (ELAND + SAMtools). Gray and black indicate confirmed mutations by Sanger sequencing and false positives, respectively. The alternate read ratios of the confirmed mutations were distributed at peak = 0.5. Possible false positives could be efficiently excluded by cutting both sides of the alternate read ratio. Dotted line represents a proposed cut-off line, i.e., 0.3 ≤ ratio ≤ 0.7.

40

0.3 30mg / 0mg

60

Fig. 5. Fold increase of the number of mutation candidates detected in the offspring of ENU-treated and control fathers in analysis 2 (BWA + GATK). Ratio of the total number of de novo mutation candidates from the ENU and control groups were plotted against minimum read depth in a Trio. The arrow indicates the peak of the fold-increase of the mutation candidates. When read depth was ≥ 27, the foldincrease was 41, 10 and 4 for 85, 30 and 10 mg/kg ENU-treated groups, respectively.

(GATK) without Sanger sequencing confirmation. The same read data (fastq files generated from 24 mice in analysis 1) were used for analysis 2. The reads were mapped by BWA onto the reference mouse genome, where 51%–72% of the mapped reads were mapped onto the exon region. Average read depth on the target was 150 (98–223 per sample). The proportion of target bases that had a depth of 10-fold or more was 91–99%. SNVs and small indels were detected by GATK for each animal. Unique de novo mutation candidates in the offspring were then scored. The ratio of the number of mutation candidates in the ENU-treated/control groups was plotted against the minimum read depth in a Trio (Fig. 5). The ratio plots peaked at read depths ≥ 27. The fold-increase at this peak was 41, 10 and 4 for 85, 30 and 10 mg/kg ENU-treated groups, respectively. This was the most sensitive setting under these experimental conditions and a read depth ≥ 27 in a Trio was selected as the cut-off value. The number of bases in the exome was calculated using the same cut-off conditions (Supplementary Fig. S3). The nucleotide sequences that passed in the exome comprised 14.8–36.3 Mb per offspring. In the ENU group,

≤ 1.0

≤ 0.9

≤ 0.95

100

≤ 0.8

80

≤ 0.85

60

≤ 0.7

40

Read depth ≥ XX

≤ 0.75

20

≤ 0.6

0

≤ 0.65

0

0

≤ 0.5

10

≤ 0.55

5

20

≤ 0.4

10

30

≤ 0.45

15

40

≤ 0.3

20

0.7

50

≤ 0.35

85mg / 0mg

25

0.2 ≤…

Fold increase

30

70

No. of mutation candidates

35

10mg / 0mg

Alternate read ratio Fig. 6. Distribution of the mutation candidates with the alternate read ratio in analysis 2 (BWA + GATK). The alternate read ratios of the confirmed mutations were distributed at peak = 0.5. Dotted line represents a cut-off line, i.e., 0.3 ≤ ratio ≤ 0.7.

219 candidate mutations were detected in 16 offspring (Table 2). For those candidates, the ratio of alternate reads to total reads was scored (Fig. 6). The alternate read ratio peaked at 0.5. The mutation candidates that had alternate read ratios of 0.3 ≤ ratio ≤ 0.7 were considered validated mutations. In order to exclude possible false mutations, we added another filter (Supplementary Table S5). In order to pass the filter, the consensus sequence from the other 23 mice had to be identical and homozygous at the mutated position. This filter excluded three mutation candidates. First, a 2 bp-deletion at a GG sequence in chromosome 8 of animal 991 was considered a false mutation because the reference sequence was 5 -TGGGGGGGGGGGGGA-3 and 13 other mice had a single G deletion and 10 mice had no mutation at this position. Second, a single T insertion in chromosome 9 in 995 was identified as a false mutation because the reference sequence was 5 -GTTTTTTTTTTTTTTTC-3 and 17 other mice had a single T deletion, 3 mice had a TT deletion, and 3 mice had no mutation at this position. The ambiguous calls might be sequencing errors in the long G- or T-run sequences. Third, an A to C mutation in chromosome 16 of animal 504 was considered a false mutation. Because all mice had T:C:A at a 2:1:1 ratio at this position, it may have been

K. Masumura et al. / Mutation Research 810 (2016) 30–39

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Table 2 Frequencies of inherited mutations in the offspring of ENU-treated fathers in analysis 2 (BWA + GATK). Average ± SD

G1 ID

P ID

M ID

No. of bases sorted in exome (depth ≥ 27)

NGS-called mutation (depth ≥ 27)

Filtered mutation

Mutation frequency (×10−8 /base)

ENU 85 mg/kg × 2 male 991 male 992 female 994 female 995

8 8 8 8

99 99 99 99 total

28,604,575 23,672,041 28,752,329 28,824,263 109,853,208

38 36 50 39 163

36 36 41 33 146

125.9 152.1 142.6 114.5 132.9

133.8 ± 16.8*

62 62 62 62 total

36,152,303 36,252,974 36,169,148 36,331,691 144,906,116

10 8 9 12 39

10 8 9 11 38

27.7 22.1 24.9 30.3 26.2

26.2 ± 3.5*

52 52 52 52 total

22,052,119 22,000,542 21,353,688 22,066,220 87,472,569

2 1 10 1 14

1 1 5 1 8

4.5 4.5 23.4 4.5 9.1

9.3 ± 9.4*

50 50 50 50 total

15,131,314 14,916,841 15,031,041 14,825,599 59,904,795

0 0 2 1 3

0 0 0 0 0

0.0 0.0 0.0 0.0 0.0

<1.7

30 mg/kg × 2 606 male male 607 female 610 female 611 10 mg/kg × 2 523 male 524 male 528 female 529 female Vehicle × 2 male 501 male 502 503 female female 504 *

44 44 44 44

29 29 29 29

43 43 43 43

Asterisks indicate significant differences versus control (p < 0.05, Steel test).

160

Mutation frequency (x 10-8 /base)

*

*

dard deviation. No significant difference was observed in the MFs between male and female offspring.

140

3.4. Mutation spectra of NGS-detected inherited germline mutations

120

100

80

60

* *

40

* 20 <4.8

<1.7

0 0 mg/kg x2

ENU 10 mg/kg x2

ENU 30 mg/kg x2

ENU 85 mg/kg x2

Fig. 7. Frequencies of inherited germline mutations estimated by whole-exome sequencing analyses. Gray is analysis 1 (ELAND + SAMtools with Sanger sequencing confirmation). Black is analysis 2 (BWA + GATK with filtering). The frequencies are presented as an average of four offspring with standard deviations. Asterisks indicate significant differences versus the control group (p < 0.05, Steel test).

caused by mis-mapped reads and coincidently called as a mutation in one mouse. The MFs were calculated by the number of filtered mutations (Table 2). The frequencies of inherited mutations in the 85, 30 and 10 mg/kg ENU-treated groups were 133.8 × 10−8 , 26.2 × 10−8 , and 9.3 × 10−8 per base, respectively. The MFs increased with dose dependency and were significantly higher in three ENU-treated groups than that in the control group (p < 0.05, Steel test). The doserelated increase of the frequencies of inherited germline mutations estimated by the two analyses was compared (Fig. 7). There was no significant difference in the MFs between analysis 1 (with confirmation by Sanger sequencing) and analysis 2 (without Sanger sequencing). In the 10 mg/kg ENU-treated group in analysis 1, the MF increase was not significant because of a relatively large stan-

Mutation spectra of the inherited germline mutations detected in the offspring by NGS analyses are summarized in Table 3 (Analysis 1) and Table 4 (Analysis 2). In both analyses, ENU preferentially induced A:T to G:C and A:T to T:A mutations as dose response. G:C to A:T mutations were also induced. These characteristics were common in both analyses and there was no significant difference in the mutation spectra in the 85 mg/kg ENU-treated group (Chi-square test). The mutations detected were randomly distributed in the exome and no mutational hot spots were observed (Supplementary Tables S4 and S5). 4. Discussion ENU-induced mutations in somatic and germ cells of the treated males and inherited offspring were investigated using transgenic mouse gene mutation assays and whole-exome sequencing analyses. The exposure to germ cells of ENU-treated mice was confirmed by a dose-dependent increase of gpt mutant frequency in sperm DNA (Fig. 2). gDNA from experiment 1 (high-dose and control) and experiment 2 (mid- and low-dose) was used for whole-exome sequencing. In analysis 1, the read data were mapped by ELAND and SNVs were called by SAMtools. A total of 87 candidates for de novo mutations were detected from 16 offspring and 90% (78/87) were confirmed as true mutations by Sanger sequencing (Table 1). This was an improvement over the 79% (126/160) identified in the previous study using a single ENU-dose [18]. One reason for this difference was sample size. The previous study used 12 mice (one treated and one control family: each family was represented by the father, mother, and four offspring). This study used 24 mice (three treated and one control family). The criteria for identifica-

36

K. Masumura et al. / Mutation Research 810 (2016) 30–39

Table 3 Mutation spectra in the offspring of ENU-treated mice in analysis 1 (ELAND + SAMtools with Sanger seq.). Control

ENU

Vehicle × 2

10 mg/kg × 2

No.

No.

%

MF (×10−8 /base)

No.

%

MF (×10−8 /base)

No.

%

MF (×10−8 /base)

Base substitution Transition 0 G:C to A:T A:T to G:C 0

1 0

50.0 0.0

3.6 0.0

7 9

33.3 42.9

9.6 12.4

10 22

18.2 40.0

22.6 49.6

Transversion G:C to T:A G:C to C:G A:T to T:A A:T to C:G

0 0 0 0

1 0 0 0

50.0 0.0 0.0 0.0

3.6 0.0 0.0 0.0

4 0 1 0

19.0 0.0 4.8 0.0

5.5 0.0 1.4 0.0

2 0 20 0

3.6 0.0 36.4 0.0

4.5 0.0 45.1 0.0

Deletion Insertion

0 0

0 0

0.0 0.0

0.0 0.0

0 0

0.0 0.0

0.0 0.0

1 0

1.8 0.0

2.3 0.0

Total

0

2

100

7.1 ±14.3 SD

21

100

28.9 ±12.3 SD

55

100

124.1 ±21.6 SD

MF (×10−8 /base)

<4.8

30 mg/kg × 2

85 mg/kg × 2

Table 4 Mutation spectra in the offspring of ENU-treated mice in analysis 2 (BWA + GATK with filtering). Control

ENU

Vehicle × 2

10 mg/kg × 2

No.%

MF (×10−8 /base)

30 mg/kg × 2

85 mg/kg × 2

No.

%

MF (×10−8 /base)

No.

%

MF (×10−8 /base)

No.

%

MF (×10−8 /base)

Base substitution Transition 0 G:C to A:T A:T to G:C 0

4 4

50.0 50.0

4.7 4.7

12 15

31.6 39.5

8.3 10.3

27 64

18.5 43.8

24.7 58.7

Transversion G:C to T:A G:C to C:G A:T to T:A A:T to C:G

0 0 0 0

0 0 0 0

0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0

4 1 5 0

10.5 2.6 13.2 0.0

2.8 0.7 3.4 0.0

11 0 37 6

7.5 0.0 25.3 4.1

10.1 0.0 33.9 5.5

Deletion Insertion

0 0

0 0

0.0 0.0

0.0 0.0

1 0

2.6 0.0

0.7 0.0

1 0

0.7 0.0

0.9 0.0

Total

0

8

100

9.3 ±9.4SD

38

100

26.2 ±3.5SD

146

100

133.8 ±16.8SD

<1.7

tion of unique de novo mutations were stricter in this study and reduced the inclusion of false mutations. In addition, the filter used in this study selected mutation candidates as those whose alternate read rate was more than 20%. It is thought that a lower alternate read ratio may not represent germline mutations, but rather somatic mosaic mutations and/or sequencing errors [30]. Inherited germline mutations could be detected as heterozygous mutations in the offspring. These results indicate that the Sanger-confirmed mutations had a distribution that was centered at an alternate read ratio = 0.5 (Fig. 4). Most false positive calls were observed at less than 0.3. This suggests that filtering the mutation candidates by low alternate read ratio is an effective method to identify true germline mutations. In analysis 2, the same read data generated from 24 mice were mapped by BWA, and SNVs were called by GATK. BWA resulted in a better mapping score than ELAND in both the percentage of the mapped reads on a target exome and the average read depth on the target. This suggests that BWA efficiently maps sequenced reads onto the reference sequence. If BWA allows more ambiguous bases to be mapped, the mapped sequences may include more variant reads. This could have resulted in more de novo mutation candidates and more bases that passed a cut-off value. A total of 219 candidates were detected from 16 offspring in analysis 2. This was 2.5-fold more than those called in analysis 1. Alternate read ratios of the mutations showed similar distributions in analysis 1

and 2 (Figs. 4 and 6). Thus, mutation candidates were simply filtered by alternate read ratios 0.3–0.7 rather than confirmation by Sanger seqencing. In addition, three candidates were filtered because they were considered sequencing errors due to long sequences of continuous bases, or caused by mis-mapped reads at multiple sites in the reference sequence. Although the numbers of mutations detected in analysis 2 was 2.5-fold higher than analysis 1, (192 and 78, respectively), the frequencies of inherited germline mutations calculated were similar (Fig. 7). This suggests that the detection methods of de novo germline mutations can work well regardless of what mapping and SNV calling tools are used, and that confirmation by Sanger sequencing can be replaced by filtering of alternate read ratios. The frequencies of inherited mutations increased with dose of ENU. These frequencies were 133.8 ± 16.8 × 10−8 per base in the 85 mg/kg ENU-treated group in analysis 2 of this study and 184 ± 48 × 10−8 in the previous study [18]. No significant difference was observed between the two studies; however, the slightly higher MF in the previous study may due to the fact that no filtering of the alternate read ratios was performed. Other ENU mutagenesis studies using a similar dosing design as our study, i.e., two or three weekly intraperitoneal injections with 75–100 mg/kg body weight, reported comparable MFs (approximately 1 × 10−6 per base) [31–33]. Therefore, the ENU-induced MF estimated by NGS

K. Masumura et al. / Mutation Research 810 (2016) 30–39

200

Mutation frequency (x 10-8 /base)

in this study is comparable to those estimated by other detection methods. ENU directly reacts with DNA and generates a variety of DNA adducts and causes base substitutions [20,34–39]. The mutation spectrum of ENU-induced inherited mutations is shown in Tables 3 and 4. Characteristic ENU-induced mutations comprised base substitutions at A:T bps, where A:T to G:C (43.8%) and A:T to T:A (25.3%) were observed in 85 mg/kg ENU-treated groups (Table 4). The mutation spectrum was reproducible in both analysis 1 and 2. It was also similar to the mutation spectrum of ENU reported in another genome-wide analysis [31]. The mutation spectra of endogenous Hprt in splenic lymphocytes of ENU-treated mice showed that A:T base substitutions were most common [40,41]; however, the most predominant type was A:T to T:A in the Hprt locus, but A:T to G:C according to exome sequencing. Interestingly, transgenic reporter gene mutation assays using lacZ, lacI, and gpt genes have demonstrated that the mutation spectra of ENU-induced somatic and germ cell mutations included base substitutions at both A:T and G:C bps, where A:T to T:A and G:C to A:T were the predominant substitutions [18]. Many factors, such as transgenic reporter genes versus exome sequences, phenotypic selection versus direct sequencing, and somatic versus inherited germline mutations could contribute to these differences in mutation spectra. In principle, direct sequencing might reflect in vivo mutagenesis better than phenotypic selection because there is no selection bias; however, NGS may also be affected by technical biases in sequencing and mutation detection methods. The control background MF could not be estimated in this study because no confirmed or filtered mutations were detected. The MF in the control group was less than 4.8 × 10−8 per base in analysis 1. It was less than 1.7 × 10−8 per base because more bases were sorted in analysis 2. In the previous study, we reported that 12 NGS-called mutation candidates among the same control offspring and three mutations were confirmed by Sanger sequencing [18]. The difference could be explained by the sorting conditions. Because the SNVs found in only a single offspring were selected as the candidates, this study analyzing 24 mice had stricter selection criteria than those in the previous study analyzing 12 mice. Another difference is that the filtering with alternate read ratio was not used in the previous study. The germline mutation rate in humans is estimated to be approximately 1 × 10−8 per base per generation [1–5]. The control germline MF in mice has been reported as 0.54 × 10−8 [42] or 0.38 × 10−8 [43]. According to the sorting conditions employed in this study, i.e., using a minimum read depth in a Trio, the number of bases sorted with the same cut-off condition per offspring in the control group comprised 5.2–5.5 Mb in analysis 1 and 14.8–15.1 Mb in analysis 2 (Tables 1 and 2). Therefore, the control MF of each animal could have been zero. Pooling the background data obtained from exome sequencing with more read depth in the untreated mice is necessary for estimating the background MF and standard deviation in the laboratory mice. Rather than exome sequencing, whole genome sequencing could be a promising solution in determining the germline MFs in background levels. In the ENU-treated male mice, the gpt mutant frequencies increased with dose in liver, testis, and sperm DNA (Fig. 2). No threshold dose was observed in the experiment because the mutant frequencies were significantly higher even in the lowest dose group

37

150

100

50

0

0

50

100

150

200

Total dose of ENU (mg/kg) Fig. 8. Dose response of the ENU-induced inherited mutations in analysis 2 (BWA + GATK with filtering). The frequencies are presented as an average of four offspring with standard deviations. An approximated straight line (R2 = 0.94) is shown.

than that of the control group. Although a relatively higher mutant frequency was observed in the liver than in the testis and sperm in the 30 mg/kg ENU-treated group, this was not reproducible in other doses. Dose-response was further analyzed in those tissues using a benchmark dose approach and a lower confidence limit of 10% benchmark response rates (BMDL10 ) was calculated using the US Environmental Protection Agency’s (EPA) Benchmark Dose Software (BMDS v2.5: https://www.epa.gov/bmds) (Table 5 and Supplementary Fig. S4). The BMDL10 values were 0.499, 0.606, and 1.344 mg/kg for sperm, liver, and testis, respectively, and were similar within a 3-fold range. This may suggest that there is no significant difference in sensitivity of the ENU-induced mutations between somatic and germline cells of mice when ENU was injected by i.p.; however, reasonable dose-response analysis requires more dose points around points of departure (PoD). It has been suggested that testes present possible blood-testis barriers to blood-borne substances, but that these barriers do not effectively block genotoxicants [11,44]. It has been reported that the BMDL for oral subchronic exposure of ENU was higher than that for acute exposure in lacZ mutant frequency in sperm of MutaTM Mouse [45]. This suggests that saturation of DNA repair affects a mutagenic mode of action. It also should be noted that the mutant frequencies may include some clonally-expanded mutations because the sampling time was approximately 20 weeks after the acute treatment in this study. If ENU-induced germ cell toxicity is followed by recovery of growth, this may cause clonal mutations that affect dose-response analyses. In the NGS analysis, a dose-dependent increase of independent mutations in the offspring of the ENU-treated fathers was observed (Fig. 8). Although only one family was analyzed in each dose and the background MF was not determined (assumed to be zero in Fig. 8), the dose-response fits a linear line well (R2 = 0.94). ENU is known as a direct-acting alkylating agent [20]. Thus, a linear dose-response relationship might be a reasonable assumption. The BMDL could not be calculated for the inherited mutations because

Table 5 BMDL10 of the ENU-induced somatic and germ cell mutations. Total ENU dose (mg/kg)

Animal/group

Tissue

Endpoint

BMD10 (mg/kg)

BMDL10 (mg/kg)

Model

0, 20, 60, 170 0, 20, 60, 170 0, 20, 60, 170

5 5 5

Liver Testis Sperm

gpt mutant frequency gpt mutant frequency gpt mutant frequency

2.719 3.727 0.946

0.606 1.344 0.499

Exponential5 Power Hill

Graphs of each dose-response model are shown in Supplemental Fig. S4.

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K. Masumura et al. / Mutation Research 810 (2016) 30–39

background MF was not determined. More points at lower doses need to be analyzed in order to establish a practical threshold and PoD. An adverse outcome pathway (AOP) for alkylation of DNA in male premeiotic germ cells that leads to heritable mutations has been developed [46]. In the concept of linkage of sequential key events in AOP, sperm mutation frequency should be equal or greater than the mutation rate measured in offspring because some mutations confer selective disadvantages and will be eliminated and thus not detected in offspring. In this study, however, the fold increases in the MFs of ENU-treated groups were higher in the offspring than in the sperm. It was difficult to demonstrate such a quantitative relationship because the frequencies of inherited mutations were estimated by direct sequencing and those in sperm were detected by bacteria-mediated phenotypic assay. If possible, the MFs in germ cells and inherited offspring should be estimated using the same NGS technology. For example, genome-wide, singlecell analysis was performed in order to measure de novo mutation rates and recombination activity in human sperm cells [47]. In this study, we demonstrated a dose-dependent increase of inherited de novo germline mutations induced by a chemical mutagen using whole-exome sequencing in 24 mice. The robustness of the detection methods was confirmed by using different tools for mapping and variant calling. We demonstrated that confirmation of mutations by Sanger sequencing could be replaced by filtering. These results suggest that direct sequencing analysis may be a powerful tool to investigate germline mutation frequency and mutation spectrum induced by environmental mutagens. Sequencing costs are dramatically decreasing, thus, this may be a promising tool for regulatory purposes. Theoretically, this approach is applicable to any species including humans and wild organisms. Rahbari et al. investigated human germline mutations by comparing multisibling families using whole genome sequencing and reported that the mutation rate increased with paternal age [48]. Inherited germline mutations could be an important endpoint for evaluating the mutagenic effects of environmental chemicals and biological perturbations. Conflict of interest statement There are no conflicts of interests. Acknowledgment This study was supported by JSPS KAKENHI Grant Number 25281027 for K.M. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mrgentox.2016. 09.009. References [1] J. Shendure, J.M. Akey, The origins, determinants, and consequences of human mutations, Science 349 (2015) 1478–1483. [2] D.F. Conrad, J.E. Keebler, M.A. DePristo, S.J. Lindsay, Y. Zhang, F. Casals, Y. Idaghdour, C.L. Hartl, C. Torroja, K.V. Garimella, M. Zilversmit, R. Cartwright, G.A. Rouleau, M. Daly, E.A. Stone, M.E. Hurles, P. Awadalla, P. Genomes, Variation in genome-wide mutation rates within and between human families, Nat. Genet. 43 (2011) 712–714. [3] A. Kong, M.L. Frigge, G. Masson, S. Besenbacher, P. Sulem, G. Magnusson, S.A. Gudjonsson, A. Sigurdsson, A. Jonasdottir, A. Jonasdottir, W.S. Wong, G. Sigurdsson, G.B. Walters, S. Steinberg, H. Helgason, G. Thorleifsson, D.F. Gudbjartsson, A. Helgason, O.T. Magnusson, U. Thorsteinsdottir, K. Stefansson, Rate of de novo mutations and the importance of father’s age to disease risk, Nature 488 (2012) 471–475.

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