Insights into DNA repeat expansions among 900,000 biobank participants

Short tandem repeats (STRs) of 1–6 bp of DNA are mutable genomic elements with diverse influences on cellular and organismal phenotypes². Common STR polymorphisms, which have been characterized in human populations using short-read³ and long-read⁴ sequencing, influence gene expression⁵ and complex traits^6,7. Rare STR expansions cause more than 60 genetic disorders¹. The allelic diversity that underlies these effects is generated by frequent mutation: around 1 million polymorphic STRs in the human genome generate around 50–60 de novo repeat-length mutations per offspring^8,9,10. Germline mutation rates of specific STRs vary widely¹¹ and are influenced by repeat motif sequence, interruptions of pure repeats and number of repeat units^8,9,10,11,12 as well as genetic variation in DNA-repair genes⁹.

STRs are also prone to somatic mutation², and lifelong somatic expansion in at least one STR locus can lead to disease. Recently, genome-wide association studies (GWASs) have provided insights into the molecular mechanisms underlying somatic repeat instability¹³ by finding common genetic modifiers of the timing or progression of Huntington’s disease (HD)^{14,15,16,17,18,19}, which is caused by inherited alleles in which a CAG repeat in the HTT gene is longer than 35 CAGs; these genetic modifiers were found in many DNA-repair genes that affect the stability of DNA repeats^{14,15,16,17,18,19}. Neurodegeneration in HD was subsequently found to be caused by somatic expansion of this repeat beyond a high threshold of about 150 CAG repeats²⁰. The genetic-modifier studies, so far of up to 16,640 persons with HD, have provided early clues toward a few potential therapeutic targets for slowing or halting somatic expansion of DNA repeats; however, the number of such potential targets is so far modest.

Whole-genome sequencing (WGS) of biobank cohorts offers opportunities to study repeat instability in much larger sample sizes than previously possible. Here we analysed repeat instability at 356,131 polymorphic repeat loci using short-read WGS data from the blood-derived DNA of 490,416 participants in UK Biobank (UKB)²¹ and 414,830 participants in All of Us (AoU)²². To do so, we developed several computational techniques, overcoming challenges in estimating the length and instability of DNA repeats from large numbers of short WGS reads²³. These methods enabled us to characterize allele-specific expansion and contraction rates of common repeats, identify genetic influences on somatic repeat expansion and identify associations of expanded repeats with diseases.

We began by analysing CAG trinucleotide repeats, which we could efficiently ascertain from biobank sequencing data and which cause many progressive, neurodegenerative repeat-expansion disorders^1,2. We identified UKB participants with long CAG-repeat alleles (≥45 repeat units) by analysing WGS data for 151 bp sequencing reads comprised entirely or almost entirely of CAG-repeat units (in-repeat reads (IRRs); Extended Data Fig. 1a). Such reads were easily extractable, as nearly all of them had been aligned to the TCF4 CAG-repeat sequence by bwa²⁴ (Supplementary Fig. 1). For each participant with one or more IRRs, we determined the locus or loci from which the IRRs originated by identifying mate sequences that mapped near one of 1,159 commonly polymorphic CAG-repeat loci³.

The vast majority of CAG-repeat expansions in the UKB occurred at only a few loci: 18 autosomal CAG-repeat sequences in the human genome were expanded to at least 45 repeat units in at least five UKB participants (Extended Data Fig. 1b and Supplementary Table 1). Three repeat loci were expanded in thousands of UKB participants—CA10 (137,673 participants), TCF4 (42,004) and ATXN8OS (7,736)—together accounting for 97% of all observed expansions beyond 45 repeat units. Most of these repeats (15 out of 18) were in transcribed genomic regions, consistent with the idea that transcription contributes to repeat instability²⁵ (Supplementary Table 2). For 9 out of the 18 repeats, expanded alleles are known to be pathogenic¹.

To study the mutability of these repeats, we measured the lengths of common, short alleles of each repeat (≤30 repeat units) by analysing sequencing reads that spanned repeat alleles, focusing on 15 repeat loci that passed additional filters (Extended Data Fig. 1 and Supplementary Table 1). These analyses recovered repeat-length distributions consistent with previous analyses²⁶ (Extended Data Fig. 1b).

We first analysed germline instability of these repeats, using the large UKB cohort to obtain high-resolution estimates of germline mutability (providing context for analyses of somatic mutability). To estimate allele-specific intergenerational expansion and contraction rates of each repeat, we analysed length discordances among alleles belonging to genomic tracts inherited identical-by-descent (IBD) from shared ancestors, building on IBD-based analyses of single-nucleotide mutations^27,28,29 (Fig. 1a). We validated this approach using two complementary methods (Supplementary Fig. 2).

a, Germline mutation rates were estimated by analysing discordance rates among alleles inherited within IBD tracts shared by pairs of UKB participants. Ancestral alleles were imputed from more-distantly shared haplotypes. b, Per-generation rates of germline expansion (+1 repeat unit) and contraction (−1 repeat unit) of GLS and TCF4 repeat alleles, estimated in the UKB. c, The analytical strategy for estimating somatic mutation rates by detecting and filtering out reads that are likely to reflect PCR artifacts introduced during sequencing. During PCR-based bridge amplification on a flow cell, a DNA fragment is clonally amplified into a cluster of colocalized DNA molecules. A PCR stutter error results in a polyclonal cluster containing a mixture of DNA molecules with and without the error. If the molecules containing the error constitute the majority of the cluster, the sequencing read generated from the cluster (reflecting the majority base at each position within the read) will contain the error, but the heterogeneity of the cluster will reduce base qualities at positions within the read that mismatch between molecules with and without the error. d, The rates of somatic expansion of GLS and TCF4 repeat alleles (that is, the fractions of blood cells in which an allele has expanded by +1 repeat unit), stratified by age in AoU. e, Somatic mutation rates in the UKB plotted against germline mutation rates for GLS and TCF4 repeat alleles. The error bars show the 95% confidence intervals (CIs). Sample sizes are provided in Supplementary Table 3.

Across all 15 CAG-repeat loci, intergenerational mutation rates increased with allele length, rising to 0.5–0.9% per generation for single-repeat-unit expansions of the longest common alleles of repeats in GLS, DMPK and ATXN8OS (Extended Data Figs. 1b and 2). The average mutation rate per locus ranged from 8.2 × 10⁻⁵ to 9.5 × 10⁻⁴ (Supplementary Table 3). These rates are relatively high for trinucleotide repeats⁸ and exceed the genome-wide average for STRs (around 5 × 10⁻⁵ per haplotype per generation)^8,9,10. Repeat loci tended to either expand more often than contract (particularly so for ATXN8OS and GLS) or to have similar expansion and contraction rates (Extended Data Figs. 1b and 2). Interruptions of repeat sequences (that is, intrarepeat sequence variants) greatly stabilized alleles: a common 18-repeat TCF4 allele containing an interruption in its ninth repeat unit exhibited a 135-fold (54–336, 95% CI) lower expansion rate compared with the uninterrupted 18-repeat allele, and an interruption in the second-to-last repeat unit of a 19-repeat GLS allele decreased the expansion rate 3.7-fold (1.9–7.2, 95% CI) (Fig. 1b). These results corroborate previous observations that repeat interruptions stabilize the expansion of pathogenic alleles^30,31,32 and quantify the strength of such effects in the germline.

These high rates of germline instability led us to wonder whether common alleles of some repeats might be sufficiently unstable in blood cells for somatic length-change mutations to be ascertainable in short-read WGS data. Identifying such mutations is challenging because polymerase slippage during PCR amplification can spuriously alter repeat lengths^33,34,35. Such ‘PCR stutter’ errors are unavoidable during Illumina sequencing by synthesis, which uses PCR for bridge amplification of DNA fragments³⁶. However, we realized that this PCR error mode tends to produce predictable patterns of reduced base quality scores within sequencing reads, enabling us to detect and exclude most reads with artefactual CAG length mutations (Fig. 1c and Supplementary Fig. 3). We applied this filtering strategy in the UKB to estimate repeat-specific, allele-specific somatic expansion rates, which we quantified as the average fraction of blood cells in which a given repeat allele has expanded by one repeat unit.

For 4 out of the 15 CAG repeats (in TCF4, GLS, DMPK and ATN1), we detected significant increases in somatic single-repeat-unit expansion rates with age (Extended Data Fig. 3). These findings were replicated in AoU, in which the wider age range of participants (aged 18 to 90+ years) revealed clear increases in fractions of blood cells containing somatic expansions with increasing age and with increasing allele length (Fig. 1d and Extended Data Fig. 4). TCF4 repeats were the most somatically unstable: individuals carrying alleles with 25 or more repeat units typically exhibited somatic expansion in more than 1% of blood cells by the age of 55 years (Fig. 1d). We did not observe age-associated contraction of any of the 15 repeat loci.

Comparing these estimates of somatic one-repeat-unit expansion rates with our estimates of intergenerational mutation rates showed that the relative (blood/germline) rates of CAG-repeat expansion varied severalfold across repeat loci (Fig. 1e). The TCF4 repeat exhibited the greatest somatic instability in blood but was relatively stable in the germline, whereas the GLS repeat displayed the opposite behaviour (Fig. 1e), as did the DMPK repeat (Extended Data Figs. 1b, 2 and 4). These results align with observations that somatic instability of pathogenic repeat expansions is highly tissue-specific, perhaps due to differences in transcription or trans-acting factors^25,37,