Clinical Genetics Group, MRC-Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
Hum Reprod. 2019 Aug 1;34(8):1404-1415. doi: 10.1093/humrep/dez090.
What effect does cancer treatment have on levels of spontaneous selfish fibroblast growth factor receptor 2 (FGFR2) point mutations in human sperm?
Chemotherapy and radiotherapy do not increase levels of spontaneous FGFR2 mutations in sperm but, unexpectedly, highly-sterilizing treatments dramatically reduce the levels of the disease-associated c.755C > G (Apert syndrome) mutation in sperm.
Cancer treatments lead to short-term increases in gross DNA damage (chromosomal abnormalities and DNA fragmentation) but the long-term effects, particularly at the single nucleotide resolution level, are poorly understood. We have exploited an ultra-sensitive assay to directly quantify point mutation levels at the FGFR2 locus.
STUDY DESIGN, SIZE, DURATION: 'Selfish' mutations are disease-associated mutations that occur spontaneously in the sperm of most men and their levels typically increase with age. Levels of mutations at c.752-755 of FGFR2 (including c.755C > G and c.755C > T associated with Apert and Crouzon syndromes, respectively) in semen post-cancer treatment from 18 men were compared to levels in pre-treatment samples from the same individuals (n = 4) or levels in previously screened population controls (n = 99).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Cancer patients were stratified into four different groups based on the treatments they received and the length of time for spermatogenesis recovery. DNA extracted from semen samples was analysed using a previously established highly sensitive assay to identify mutations at positions c.752-755 of FGFR2. Five to ten micrograms of semen genomic DNA was spiked with internal controls for quantification purposes, digested with MboI restriction enzyme and gel extracted. Following PCR amplification, further MboI digestion and a nested PCR with barcoding primers, samples were sequenced on Illumina MiSeq. Mutation levels were determined relative to the spiked internal control; in individuals heterozygous for a nearby common single nucleotide polymorphism (SNP), mutations were phased to their respective alleles.
Patients treated with moderately-sterilizing alkylating regimens and who recovered spermatogenesis within <3 years after therapy (Group 3, n = 4) or non - alkylating chemotherapy and/or low gonadal radiation doses (Group 1, n = 4) had mutation levels similar to untreated controls. However, patients who had highly-sterilizing alkylating treatments (i.e. >5 years to spermatogenesis recovery) (Group 2, n = 7) or pelvic radiotherapy (Group 4, n = 3) exhibited c.755C > G mutation levels at or below background. Two patients (A and B) treated with highly-sterilizing alkylating agents demonstrated a clear reduction from pre-treatment levels; however pre-treatment samples were not available for the other patients with low mutation levels. Therefore, although based on their age we would expect detectable levels of mutations, we cannot exclude the possibility that these patients also had low mutation levels pre-treatment. In three patients with low c.755C > G levels at the first timepoint post-treatment, we observed increasing mutation levels over time. For two such patients we could phase the mutation to a nearby polymorphism (SNP) and determine that the mutation counts likely originated from a single or a small number of mutational events.
LIMITATIONS, REASONS FOR CAUTION: This study was limited to 18 patients with different treatment regimens; for nine of the 18 patients, samples from only one timepoint were available. Only 12 different de novo substitutions at the FGFR2 c.752-755 locus were assessed, two of which are known to be disease associated.
Our data add to the body of evidence from epidemiological studies and experimental data in humans suggesting that male germline stem cells are resilient to the accumulation of spontaneous mutations. Collectively, these data should provide physicians and health-care professionals with reassuring experimental-based evidence for counselling of male cancer patients contemplating their reproductive options several years after treatment.
STUDY FUNDING/COMPETING INTEREST(S): This work was primarily supported by grants from the Wellcome (grant 091182 to AG and AOMW; grant 102 731 to AOMW), the University of Oxford Medical Sciences Division Internal Fund (grant 0005128 to GJM and AG), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme (to AG) and the US National Institutes of Health (to MLM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. None of the authors has any conflicts of interest to declare.
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癌症治疗对人类精子中自发的自私性成纤维细胞生长因子受体 2（FGFR2）点突变的水平有什么影响？
化疗和放疗不会增加精子中自发 FGFR2 突变的水平，但出乎意料的是，高度致不育的治疗方法会显著降低精子中与疾病相关的 c.755C>G（Apert 综合征）突变的水平。
癌症治疗会导致短期的大量 DNA 损伤（染色体异常和 DNA 碎片化），但长期影响，特别是在单核苷酸分辨率水平上，知之甚少。我们利用一种超灵敏的检测方法，直接定量 FGFR2 基因座上的点突变水平。
研究设计、规模、持续时间：“自私”突变是指大多数男性精子中自发出现的与疾病相关的突变，其水平通常随着年龄的增长而增加。比较了 18 名男性癌症治疗后精液中 FGFR2 的 c.752-755 位（包括与 Apert 和 Crouzon 综合征相关的 c.755C>G 和 c.755C>T）突变水平与同一患者（n=4）或先前筛选的人群对照（n=99）的治疗前样本中的水平。
参与者/材料、设置、方法：根据他们接受的治疗方法和精子发生恢复的时间长短，将癌症患者分为四组。从精液样本中提取 DNA，使用先前建立的超灵敏检测方法，鉴定 FGFR2 的 c.752-755 位的突变。将 5 到 10 微克的精液基因组 DNA 与内部对照一起进行定量分析，用 MboI 限制性内切酶消化，凝胶提取。在进一步的 MboI 消化和带有条形码引物的嵌套 PCR 之后，将样品在 Illumina MiSeq 上进行测序。突变水平相对于内参进行确定；对于附近常见单核苷酸多态性（SNP）杂合的个体，突变被相位到各自的等位基因。
接受中度致不育的烷化剂治疗且治疗后 3 年内精子发生恢复的患者（第 3 组，n=4）或接受非烷化化疗和/或低性腺辐射剂量的患者（第 1 组，n=4）的突变水平与未治疗的对照组相似。然而，接受高度致不育的烷化剂治疗（即恢复精子发生需要 >5 年）的患者（第 2 组，n=7）或骨盆放疗的患者（第 4 组，n=3）的 c.755C>G 突变水平处于或低于背景水平。两名接受高度致不育的烷化剂治疗的患者（A 和 B）表现出从治疗前水平明显降低的趋势；然而，对于其他突变水平较低的患者，我们无法获得治疗前的样本。因此，尽管根据他们的年龄，我们预计会检测到可检测水平的突变，但我们不能排除这些患者在治疗前也有较低的突变水平的可能性。在三个治疗后第一个时间点 c.755C>G 水平较低的患者中，我们观察到随着时间的推移突变水平增加。对于其中两个这样的患者，我们可以将突变相位到附近的一个多态性（SNP）上，并确定突变计数可能来自一个或少数几个突变事件。
局限性、谨慎的原因：本研究仅涉及 18 名接受不同治疗方案的患者，其中 9 名患者只有一个时间点的样本。仅评估了 FGFR2 c.752-755 位的 12 个不同的新生取代，其中两个已知与疾病相关。
我们的数据增加了来自流行病学研究和人类实验数据的证据，表明男性生殖干细胞对自发突变的积累具有弹性。这些数据应共同为男性癌症患者在治疗后几年考虑其生殖选择时，为医生和医疗保健专业人员提供基于实验的令人放心的证据。
研究资金/利益冲突：这项工作主要由惠康基金会（091182 号赠款给 AG 和 AOMW；102731 号赠款给 AOMW）、牛津大学医学科学系内部基金（0005128 号赠款给 GJM 和 AG）、英国国家卫生研究院牛津生物医学研究中心计划（赠款给 AG）和美国国立卫生研究院（赠款给 MLM）资助。资助者在研究设计、数据收集和分析、决定发表或准备手稿方面没有任何作用。没有作者有任何利益冲突需要申报。
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