Attempt to Detect Long-term Exposure-induced Mutations in Trace Radiation Based on Whole-genome Decoding
Y. Gondo (Tokai University School of Medicine, Japan)
Natural mutation detection and analysis for single base substitution (SNV) using whole genome sequencing (WGS) based on the ultrafast sequencing method is advancing. The mutation rate in humans is reported to be 1.2 × 10-8/bp/generation. In the case of a human having 3 × 109 bp diploid genomes, it works out to an average of 72 mutations, not found in either parent, naturally developing in any children. As for mice, the genomes after accumulation of mutations through sib mating have been decoded and analyzed, and the rate of naturally developing SNV mutations is reported to be 5.4 × 10-9/bp/generation, which is approximately 1/2 of that for humans. We also obtained a result with extremely high reproducibility at 5.5 × 10-9/bp/generation using the complete remote crossing method for mice.
Accordingly, a trans-generational risk assessment using mice continuously exposed to low-dose radiation at approximately 50 mGy/year for several generations (lifelong-exposed mice) has been attempted using the WGS approach. The first key to this is producing and analyzing a non-irradiated control group to be compared with under the same conditions. Therefore, first, a pair of male and female mice was mated to breed. Since all infant mice are derived from these two individuals regardless of whether they are in the exposed group or the control group, we call this method expanded trio analysis. The next-generation offspring produced from these male and female mice were divided into two groups, a lifelong-exposed mouse group and a control group, for further mating and breeding. With this lifelong-exposed mouse group, exposure at 50 mGy/year is started immediately after mating. In single-dose irradiation at the several Gy level, which has previously been conducted, the mice used become temporarily infertile. Therefore, normally, recovery of their fertility is awaited before having them mate, and then risk assessment is conducted in the next generation. By contrast, if the mice are exposed at a radiation dose rate of approximately 50 mGy/year, they will not become temporarily infertile, and it is possible to start exposure immediately after mating, and constantly expose them for many generations through steps from conception to fetal period, birthing, weaning, and then sexual maturity for further mating. In addition, since all individuals are derived from the first pair of male and female mice, it is unnecessary to individually decode the parental genomes.
With the present WGS approach, sequencing is performed at large scale and high speed with a short lead of approximately 100 bp to 200 bp, and, therefore, it is impossible to detect mutations from repeated sequences, which account for approximately half of the genomes. In our analysis, the actually detected mutations were those developing in the unique sequence region, which represents 43% of the mouse genomes. In other words, the number of newly developing natural mutations that can actually be detected from one infant mouse with the WGS approach is 14.4 on average. Therefore, our plan is to first compare 10 lifelong-exposed offspring mice with 10 control offspring mice produced under the same conditions. From the control group, 144 SNVs are obtained, and this number is large enough to verify it by comparing it with the number of mutations detected from the lifelong-exposed group. Since lifelong exposure is continued in cases where a higher resolution is required, it is possible to enhance the scale of analysis by performing an additional analysis at any time. In addition, since the WGS approach is adopted, any change in the base sequence of the detected mutation can also be identified. In other words, not only a quantitative difference, but also a qualitative difference in the mutations detected in the exposed group and the control group, can be analyzed.
There are currently only few detailed analyses or data on the biological effects of low-dose long-term exposure. The primary purpose of this study is to provide a scientific ground that leads to safety and security.
The present WGS approach is performed with a short lead of approximately 100 bp to 200 bp, which is the detection limit of large-scale, high-speed sequencers. Therefore, analyses of minor mutations, such as single base substitution or deletion/insertion of several bases, are proceeding in advance. It remains a critical challenge to analyze major structural variations (SVs) at several kb or more on a large scale not only in variations induced by radiation, but also in variations caused by human diseases. We have preserved full bodies of all mice in which natural mutations are accumulated and SNV detection is performed. We have also successfully detected a large number of SV candidate sites such as deletion candidates exceeding several kb or translocations and inversions by conducting decoding using PacBio sequencing data, with which large-scale decoding of genome DNAs exceeding 10 kb can be performed, using frozen preserved mice in which SNV detection has been completed. Similarly, we also freeze-preserve the whole body of all individuals of the lifelong-exposed mice and the control group, which are obtained from this study. Currently, innovation toward high-accuracy, high-speed detection of SVs has been advancing. Concerning such new cutting-edge technologies, it is expected that SV mutation analysis exceeding several kb will be accelerated in a synergistic manner while comparing it with the existing analysis methods using the same mice for consideration.
全ゲノム解読に基づく微量放射線長期被ばく継世代誘発変異検出の試み
権藤 洋一 (Tokai University School of Medicine, Japan)
超高速シーケンシング法による全ゲノム解読（WGS）を用いた１塩基置換(SNV)の自然変異検出解析が進んでいる。ヒトでは1.2×10-8/bp/世代と報告されており、3×109bpの二倍体ゲノムをもつヒトの場合、どちらの親にもなかった変異がどの子にも平均 72 個新たに自然に生じている計算になる。マウスでも兄妹交配で変異を蓄積したのち WGS 法で解読解析し、自然に生じるSNV 変異率がヒトの約1/2の5.4×10-9/bp/世代と報告されている。われわれもマウス完全遠縁交配法を用いて 5.5×10-9/bp/世代と極めて再現性が高い結果を得ている。
そこで、WGS法を用いて50mGy/年程度の低線量放射線に数世代連続して被ばくさせたマウス（生涯被ばくマウス）を用いた継世代リスク評価を試みている。まず鍵となるのは比較対象となる非照射コントロール群を同条件で産出解析することである。そこで、まず、1雌雄ペアを交配し増殖した。すべての仔マウスは被ばく群、コントロール群を問わず、すべてこの２個体に由来するので、拡張トリオ解析と呼んでいる。この雌雄から得られた次世代産仔を、生涯被ばくマウス群とコントロールの2グループに分けさらに交配増殖する。そして、この生涯被ばくマウス群では交配直後から50mGy/年の被ばくを開始する。従来から実施されている数Gyレベルの単回照射では、マウスが一時的に不妊となるため妊性の快復を待って交配し次世代におけるリスク評価を通常行なう。一方、50mGy/年程度の放射線量率で被ばくさせた場合、一時的に不妊になることもなく、被ばくを交配直後から開始し、受精、胎児期、出産、離乳、そして性成熟してさらなる交配と、世代を越えて何世代でも常時被ばくさせることが可能となる。またすべての個体は最初の雌雄 1 ペアに由来するので、個別に親ゲノムを解読する必要もない。
現在のWGSでは、100bpから200bp 程度のショートリードで大規模高速に解読するため、ゲノムの約半分を占める反復配列などからは変異検出が不可能となっている。われわれの解析でも、実際に変異が検出できたのは、マウスゲノムの43%を占めるユニーク配列領域に生じた変異であった。すなわち、WGS法で１仔マウスから実際に検出できる新たに生じた自然変異数は平均14.4個となる。そこで、まず生涯被ばくマウス産仔10匹を同条件でえたコントロール産仔 10 匹と比較する計画で進めている。コントロール群からは144個のSNVが得られ、生涯被ばく群から検出される変異数と比較検証するには十分な数であり、解像度がさらに必要な場合には生涯被ばくを継続しているためいつでも追加解析して解析規模を拡充することが可能となる。また、WGS 法を用いているため、検出された変異の塩基配列の変化まで同定できる。すなわち、量的な違いだけでなく、被ばく群とコントロール群で検出された変異の質的な違いも解析できる。
現在、低線量長期被ばくがもたらす生物学的影響については詳細な解析やデータがほとんどなく、安全・安心につながる科学的根拠を提供することを本研究の第一の目的としている。現在のWGS法は100bp〜200bp程度のショートリード大規模高速シーケンサーの検出限界のため、１塩基置換や数塩基程度の欠失挿入といった小さな変異の解析が先行している。放射線による誘発変異に限らず、ヒト疾患原因変異においても、数 kb 以上の大きな構造変異（SV）の大規模解析が重要な課題として残されている。われわれは、これまで自然変異を蓄積しSNV検出を行なったマウスはすべて全ボディを凍結保存している。すでに、SNV 検出が完了した凍結保存マウスを用いて、10kb 超えるゲノム DNA の大規模解読が可能な PacBio シーケンシングデータを用いて解読し、数 kb を超える欠失候補や転座逆位といった SV候補箇所を多数検出することにも成功している。本研究から得られる生涯被ばくマウスおよびコントロール群も同様に全ての個体を丸ごと凍結保存する。現在、SV の高精度高速検出に向けた技術革新も進んでおり、そういった新たな最先端技術についても、同じマウスを用いて既存の解析方法との比較検討もしながら、数kbを超える SV 変異解析が、今後、相乗的に加速されていくと期待できる。
