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Somatic Point Mutations Occurring Early in Development

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Somatic Point Mutations Occurring Early in Development Downloaded from jmg.bmj.com on November 6, 2013 - Published by group.bmj.com JMG Online First, published on October 11, 2013 as 10.1136/jmedgenet-2013-101712 Somatic mosaicism ORIGINAL ARTICLE Somatic point mutations occurring early in development: a monozygotic twin study Rui Li,1,2 Alexandre Montpetit,3 Marylène Rousseau,4 Si Yu Margaret Wu,4 Celia M T Greenwood,2,5,6 Timothy D Spector,7 Michael Pollak,8 Constantin Polychronakos,4 J Brent Richards1,2,7 ▸ Additional material is ABSTRACT Indeed, somatic mutations have previously been published online only. To view The identification of somatic driver mutations in cancer demonstrated to cause non-malignant disease. Rapid please visit the journal online (http://dx.doi.org/10.1136/ has enabled therapeutic advances by identifying drug advances in molecular genetics have demonstrated jmedgenet-2013-101712). targets critical to disease causation. However, such the importance of somatic mutation in a great variety 1 genomic discoveries in oncology have not translated into of human diseases other than cancer (reviewed else- Departments of Medicine, 2 Human Genetics, Epidemiology advances for non-cancerous disease since point where). For instance, a recent proof-of-concept and Biostatistics, McGill mutations in a single cell would be unlikely to cause study has revealed the existence of early embryonic University, Montreal, Quebec, non-malignant disease. An exception to this would occur somatic mutations causing Dravet syndrome,3 as well Canada fi AKT1 2 if the mutation happened early enough in development as another novel nding of mosaic mutation in Lady Davis Institute, Jewish ’ 4 General Hospital, McGill to be present in a large percentage of a tissue s cellular a Proteus syndrome patient. A more recent publica- University, Montreal, Quebec, population. We sought to identify the existence of tion identified somatic mutations in individuals with Canada somatic mutations occurring early in human development clonal haematopoiesis but without haematopoietic 3 McGill University and Genome by ascertaining base-pair mutations present in one of a malignancies.5 Quebec Innovation Centre, pair of monozygotic twins, but absent from the other In addition, the identification of early somatic Montreal, Quebec, Canada 4Departments of Pediatrics and and assessing evidence for mosaicism. To do so, we mutations would provide preliminary insights into Human Genetics, McGill genome-wide genotyped 66 apparently healthy somatic mutation rates, which have been previously – University, Montreal, Quebec, monozygotic adult twins at 506 786 high-quality single estimated in in vitro cell models6 10 or disease– Canada nucleotide polymorphisms (SNPs) in white blood cells. gene data.11 12 Recent advances in sequencing tech- 5Department of Epidemiology, Discrepant SNPs were verified by Sanger sequencing and nology have provided such rates in tumour Biostatistics and Occupational 13–16 Health, McGill University, a selected subset was tested for mosaicism by targeted samples, and genotyping arrays have led to the Montreal, Quebec, Canada high-depth next-generation sequencing (20 000-fold detection of copy number variation in population 6 – Department of Oncology, coverage) as a surrogate marker of timing of the studies.17 19 However, the burden of point muta- McGill University, Montreal, mutation. Two de novo somatic mutations were tions in non-malignant tissue is not known, and Quebec, Canada fi 7Twin Research and Genetic unequivocally con rmed to be present in white blood further we are not aware of previous reports identi- −7 Epidemiology, King’s College cells, resulting in a frequency of 1.2×10 mutations per fying the existence of somatic point mutations in London, London, UK nucleotide. There was little evidence of mosaicism on otherwise apparently healthy individuals. 8 Departments of Medicine and high-depth next-generation sequencing, suggesting that Monozygotic twins provide a natural experiment Oncology, McGill University, Montreal, Quebec, Canada these mutations occurred early in embryonic to address these questions since any differences development. These findings provide direct evidence that between monozygotic co-twins would arise due to Correspondence to early somatic point mutations do occur and can lead to somatic changes. In the current study, we genome- Dr Brent Richards, differences in genomes between otherwise identical wide genotyped 66 monozygotic twins (33 pairs) Departments of Medicine, twins, suggesting a considerable burden of somatic to identify somatic point mutations. In addition, we Human Genetics, Epidemiology and Biostatistics, mcGill mutations among the trillions of mitoses that occur over sought validation of the candidate somatic muta- university, Montreal, Quebec, the human lifespan. tions through Sanger sequencing. Given that muta- Canada H3A 2T5; tions occurring later in organ development would [email protected] lead to somatic mosaicism within a cell line, we fi Received 3 April 2013 assessed the degree of mosaicism of identi ed Revised 17 September 2013 INTRODUCTION mutations using next-generation sequencing. Accepted 18 September 2013 Mutation is an important source of genetic vari- Together, these data provide direct evidence of the ation in the human genome. It can introduce dele- existence of somatic mutations occurring early in terious nucleotide changes to genes or provide fuel human development and a preliminary estimate of for phenotypic evolution. It is well accepted that the rate at which they occur in an otherwise 1 such mutations may lead to cancer, but it is normal population. unlikely that all base-pair mutations would cause malignancy. Rather, such stochastic events could also lead to possible disruption of an organ’s func- RESULTS tion, particularly so if the mutation were present in Data generation To cite: Li R, Montpetit A, a large proportion of the cells within a tissue. Such By genome-wide genotyping with Illumina 610K Rousseau M, et al. J Med Genet Published Online First: mutations could exist if they were introduced early single nucleotide polymorphism (SNP) arrays, in [please include Day Month in embryogenesis and their identification could the same laboratory at the same time, we obtained Year] doi:10.1136/ identify important control points in disease aeti- genotype calls on 506 821 SNPs in 33 pairs of jmedgenet-2013-101712 ology, such as has happened for malignant disease. monozygotic twins (66 individuals) after quality Li R,Copyrightet al. J Med Genet Article2013; 0author:1–7. doi:10.1136/jmedgenet-2013-101712 (or their employer) 2013. Produced by BMJ Publishing Group Ltd under licence. 1 Somatic mosaicism control (figure 1). We only included high-quality (per SNP ∼20 000-fold coverage per amplified region (see online supple- − missing rate <0.01, Hardy–Weinberg Equilibrium p>10 6) mentary table S1). common SNPs (minor allele frequency >0.05). The cut-off of Aligning the sequencing reads to the human reference genome genotyping call rate per sample was 5%. (build 37), the two somatic mutations identified by Sanger The call rate in the 66 individuals included in this study was sequencing were confirmed. The reference allele over all alleles >99.9%. Zygosity of the twins was determined by standardised ratio (RAF), as determined by allelic reads, was 1 for the homozy- questionnaire and confirmed by genotype concordance on a gous twin while the RAF was 0.5 and 0.6 for each of the hetero- genome-wide level (>99.9%) in the 33 pairs of twins. To iden- zygous twin, respectively (table 1). The reads counted prior to tify potential somatic mutations, we first assessed the genome- PCR duplicates removal gave the same ratio as that subjected to wide genotypes and identified 1087 discordant SNPs across all duplicates removal. We did not identify additional evidence for twin pairs. Of these, 1019 were present once across all twin somatic mutations from the 18 other candidate mutant single pairs, 64 were present in two pairs of twins and 4 SNPs were nucleotide variants (SNVs) (see online supplementary table S1). discordant in three pairs of twins. Excluding these four SNPs as a potential source of genotyping error (since some SNPs are Preliminary measurement of somatic mutation frequency more difficult to genotype than others), we observed a median The total number of sites investigated was determined from the of 10 discordant SNPs per pair of monozygotic twins ranging intersection of high-quality SNPs called in both co-twins that from 1 to 282 (mean=34.76). met the quality control criteria as shown in online supplemen- tary table S2. The somatic mutation frequency was calculated as Raw data inspection to identify candidate somatic the number of mutation events divided by the sum of total mutations for external verification number of SNP sites in each twin pair. The estimated mutation − Despite precautions to select only the highest quality SNPs, the frequency was 1.2×10 7 per base pair. majority of the discordant SNPs were likely to be due to geno- We observed little evidence for striking mosaicism from the typing errors. To resolve this possible source of error, we visu- next-generation sequencing results based on RAF (see online ally inspected the cluster plots of the 1087 discordant SNPs supplementary table S1). Since we observed an RAF of 0.5–0.6 between 66 monozygotic twins (33 pairs). Examination of the for the heterozygous twin at mutant sites, it is highly probable data (cluster separation, tightness, intensity and outsider geno- that the mutations we observed
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