Microsatellite Interruptions Stabilize Primate Genomes and Exist as Population-Specific Single Nucleotide Polymorphisms within Individual Human Genomes
[['Over 3% of the human genome consists of microsatellites , defined as short tandem repeats of 1–6 bases per motif unit , interspersed throughout the genome [1] .', 'Strand slippage during DNA synthesis is facilitated by the presence of tandem repeats , and has been proposed to be the dominant mutational mechanism for microsatellites [2] , [3] .', 'Perfect microsatellites contain repeats of a single motif sequence , whereas interrupted microsatellites ( iMSs ) include tandem repeats of a single motif interrupted by other bases .', 'Many microsatellites are located within coding and regulatory sequences [4] , and can be important modifiers of gene expression , affecting transcription rate , RNA stability , splicing efficiency , and RNA-protein interactions [5]–[7] .', 'Because microsatellite alleles are highly polymorphic , they may provide a large pool of heritable , phenotypic variants for subsequent selection [8]–[10] .', 'Length variation at certain microsatellites contributes to natural variation in brain development and behavioral traits [11] , and may modulate neurodegenerative disease risk [12] .', 'Microsatellite interruptions also are known to have important consequences for human health and disease .', 'For instance , germline interruptions of disease-causing microsatellite alleles act as a disease modifier for spinocerebellar ataxia type 10 [13] , and alter the age of onset of spinocerebellar ataxia type 1 [14] .', 'Importantly , the presence of interrupted alleles at the FMR gene ( Fragile X syndrome ) microsatellite diminishes the likelihood of repeat-expansion to disease length alleles in the next generation [15] , [16] .', 'Similarly , the presence of multiple interruptions at the DM-1 gene microsatellite decreases the probability of both germline and somatic expansions [17] , [18] .', 'Furthermore , a population-specific , single nucleotide polymorphism within the APC gene coding region converts an iMS ( AAATAAAA ) to a perfect microsatellite ( A ) 8 , leading to an increased risk of somatic APC mutation and colorectal cancer in Ashkenazi Jews [19] .', 'Biomedical interest in microsatellite interruptions has been renewed recently by the demonstration that iMSs within the ATXN2 ( SCA2 ) gene are associated with a different disease presentation than perfect expanded alleles [20] .', 'These studies demonstrate that a complex relationship exists between microsatellites and disease , that involves not only length but also sequence polymorphisms .', 'Importantly , iMSs might represent a reservoir of mutable alleles that can expand in subsequent generations , as was shown for SCA2 [21] and myotonic dystrophy type 2 [22] .', 'Microsatellite interruptions are major contributors to the microsatellite life cycle .', 'According to the life cycle hypothesis , a microsatellite locus undergoes stages of birth , adulthood and death during its evolution [23] .', 'Microsatellites are “born” from short tandem repeats ( proto-microsatellites ) when they reach a threshold length that alters their mutational behavior [24] , [25] .', 'Microsatellites display a characteristically high frequency of motif-based insertion/deletion ( indel ) mutations that drive high germline microsatellite mutation rates; this is in contrast to proto-microsatellites that have lower indel mutation frequencies than microsatellites [25] , [26] .', 'Microsatellites “die” when the length of the tandem repeat falls below the threshold , and interruptions are the major cause of microsatellite death [27] , [28] .', 'Some interruptions can persist for millions of years ( MYs ) , e . g . , for 19–35 MYs at one locus studied in artiodactyls [29] .', 'These features can serve as an advantage when using iMSs as markers in population genetics , since interrupted repeats exhibit lower homoplasy than uninterrupted MSs .', 'Indeed , for iMSs , the probability of acquiring an interruption by two independent events ( i . e . the probability of a homoplasy ) is much lower than the probability of inheriting this interruption from a common ancestor .', 'Because of this , iMSs might be more appropriate markers than perfect microsatellites for studying population differentiation [30] .', 'Interrupted microsatellites are more stable genetically ( less mutable , but still polymorphic ) than perfect repeats in natural chicken populations [31] , and interruptions can reduce the mutability of specific microsatellite sequences [32]–[34] .', 'However , the quantitative effects of interruptions on decreasing human microsatellite mutability have never been evaluated previously in a genome-wide study .', 'The significant role of iMSs in modifying the clinical manifestations of disease and their important contributions to genome evolution warrant a detailed understanding of iMSs .', 'Specifically , the architecture of human genomes with regard to iMSs has not been previously investigated , and the mechanism by which interruptions arise has not been extensively studied .', 'We used a multi-disciplinary approach combining computational and biochemical methods to address three biologically important questions regarding microsatellite interruptions .', 'First , what is the quantitative effect of microsatellite interruptions on microsatellite mutability genome-wide ?', 'Second , how common are microsatellite interruptions within the human genome , where do they occur , and how often are human populations polymorphic for the presence/absence of interruptions ?', 'Third , what are the possible biochemical pathways giving rise to microsatellite interruptions ?', 'Our results reveal the highly dynamic nature of microsatellite mutagenesis in the human genome , one that includes a robust level of interruption variation , and demonstrate that iMSs provide a source of population-specific genetic modifiers potentially affecting the stability of individual human genomes .'], ['To understand the impact of microsatellite interruptions on human genome stability , we first set out to determine the genome-wide magnitude of microsatellite mutability reduction due to the presence of interruptions .', 'For this analysis , we studied high-quality primate genome alignments using a comparative genomics approach .', 'Mono- , di- , tri- and tetranucleotide microsatellites above the threshold repeat number were identified in human , chimpanzee , orangutan , macaque , and marmoset reference genomes ( Table S1; penta- and hexanucleotide microsatellites were omitted due to their lower abundance and algorithmic difficulties in specifying all possible interruptions ) .', 'iMSs were identified as microsatellites in which at least one perfect repeat stretch extended beyond the threshold repeat number .', "An interruption was required to be shorter than or equal to the microsatellite's motif size .", 'For each of the five primate genomes examined , iMSs were more abundant than perfect microsatellites ( Table S1 ) .', 'When only orthologous iMSs with one or two interruptions were considered ( see Materials and Methods for details ) , iMSs numbered from 6 , 000–38 , 000 , while perfect microsatellites numbered from 8 , 000–48 , 000 , depending on the primate genome analyzed .', 'The mutability , or the average squared difference in repeat number ( allele length ) between two species [35] , was contrasted for all perfect versus interrupted microsatellites present in human-chimpanzee genomic alignments .', 'Namely , we performed a genome-wide comparison of the mutability of microsatellites with the same repeated motif that were perfect in both human and chimpanzee to that of microsatellites that were interrupted ( with the same interruption ( s ) ) in both of these species .', 'For microsatellites of all motif sizes examined , short microsatellites with one interruption were less mutable than perfect microsatellites with the same overall repeat number ( Figure 1A ) .', 'The average , genome-wide mutability difference for mononucleotides was ∼two-fold at 12 repeat units , and up to ∼six-fold for di- , tri- , and tetranucleotide microsatellites with 6 , 5 , and 4 units , respectively .', 'Microsatellites with two interruptions were , on average , one to two orders of magnitude more stable than uninterrupted microsatellites with the same repeat number ( Figure 1A ) .', 'The mutability difference between perfect and iMS loci was highest at shorter repeat numbers for all motifs .', 'Thus , the quantitative effect of a single interruption on an individual microsatellite locus can be substantial .', 'For example , more centrally located interruptions have a strong effect on mutability , dramatically lowering microsatellite mutability up to two to three orders of magnitude , whereas interruptions located on the microsatellite fringes have only a marginal effect ( Figure 1B ) .', 'The identity of the interrupting base has a non-significant effect on mononucleotide microsatellite mutability ( Figure S1 ) .', 'Armed with the knowledge that interruptions significantly stabilize microsatellites genome-wide , we next examined individual human genome microsatellites for the presence of interruption polymorphisms .', 'We found such polymorphisms to be highly abundant and informative for predicting population-specific microsatellite stabilization .', 'In this analysis , we identified 1 , 814 , 151 perfect mono- , di- , tri- , and tetra-nucleotide microsatellites above the threshold length within the reference human genome ( UCSC build hg19 ) [25] .', 'Here , we imposed an upper limit on the microsatellite lengths analyzed ( 10 , 9 , 8 , and 7 units for mono- , di- , tri- and tetranucleotide repeats , respectively ) , because we found next generation sequencing data at longer repeats to be biased due to sequencing errors and/or read-length limitations [25] .', 'For microsatellites that are perfect in the reference genome , we analyzed the frequency of iMSs within four human population groups ( African , European , East Asian , and American ) , using the 1000 Genomes Phase-1 variant call set [36] .', 'Interruptions were defined as single nucleotide polymorphisms ( SNPs ) or indels leading to a sequence within the microsatellite that differs from the full motif unit .', 'All indel and SNP variants ( with allele frequency ≥0 . 05 ) were identified , and considered to be interruptions if they were located within a microsatellite but not at the starting/ending repeat unit .', 'In this manner , we identified ∼26 , 000–40 , 000 polymorphic iMSs , depending on the population group ( Table 1 , Figure 2A; Datasets S1 , S2 , S3 , S4 , S5 ) .', 'A substantial number of interrupted alleles were present in all four population groups with different allele frequencies , corresponding to a fixation index ( FST ) of 0 . 061 ( range: 0 . 000–0 . 590; sd: 0 . 062; median: 0 . 041 ) , which falls well within the range of SNP FST values ( 0 . 052–0 . 083 ) derived from pair-wise population comparisons of the 1000 Genomes Phase-1 project [36] ( Dataset S6 ) .', 'Despite such low observed average level of population differentiation , numerous interruptions were shared by two or three population groups , or unique to a single population group ( referred to as ‘population-specific’ interruptions henceforth ) ( Figure 2A ) .', 'The greater number of interruptions within Africans compared to other population groups is likely due to a higher number of the 1000 Genomes variants in Africans , reflecting their high diversity [36] , [37] .', 'We also identified genes that encode polymorphic exonic iMSs .', 'Among the four population groups studied , ∼3 , 000–4 , 000 genes contained polymorphic interruptions within exonic microsatellites ( Table 2 ) .', 'Several genes encoding exonic iMS alleles are specific to only one population , or are shared by two or three populations ( Figure 2B; Dataset S1 , S2 , S3 , S4 , S5 ) .', 'These data demonstrate that iMSs can provide an abundant source of population-specific alleles potentially stabilizing individual genomes by lowering microsatellite mutation rates .', 'We performed more in-depth analyses of the polymorphic exonic iMSs identified above in the four human population groups to determine the potential functional impact of iMS presence on genome function .', 'Only a few of the iMSs identified are predicted to cause frameshifts or nonsynonymous mutations ( Figure S2 , Table S2 ) ; the vast majority of population-specific interruptions are not expected to alter protein sequence .', 'Thus , the primary effect of iMS may be to modulate the mutation rate of the underlying microsatellite .', 'To gain further insight into the potential biological relevance of the iMSs , we performed Gene Ontology ( GO ) analyses for each set of genes encoding population-specific iMS alleles .', 'The significantly ( p<0 . 01 ) enriched GO terms are distinct for each population .', 'For example , the GO terms enriched in the African-specific iMS genes included several neurological and organ development terms ( Table S3 ) , while those for the European-specific iMS genes were predominantly immunological terms ( Table S4 ) .', 'Since the GO vocabularies are structured such that they can be queried at different levels , we examined the smallest sized GO terms , identified the associated genes containing the iMS , and queried these genes for clinical associations using Online Mendelian Inheritance in Man ( www . omim . org ) .', 'Several genes that we identified in this manner are associated with familial disease or disease susceptibility ( Table 3 ) .', "For example , we discovered three , African-specific interrupted mononucleotide microsatellites within the HTT ( Huntington's ) gene , which correspond to perfect microsatellites in European , Asian and American populations ( Table 3 ) .", 'It is important to bear in mind that although the genes identified by this analysis are implicated in disease , the associated microsatellites have not been shown to play a causal role .', 'Therefore , these iMSs will have to be studied further for their potential role in modulating disease risk .', 'We also examined polymorphisms in 15 genes containing exonic ( coding and UTR ) iMS alleles that are well known to be associated with microsatellite expansion diseases [38] .', 'Eight loci ( ARX , CBFA1 , FMR1 , FMR2 , HOXA13 , OPMD , SCA3 , and ZIC2 ) contained no differences in microsatellite sequence from the reference genome in any of the four population groups studied .', 'Four genes ( AIB1 , SCA2/ATXN2 , SCA17 , and HOXD13 ) contained iMS alleles that differed from the reference genome sequence , and the variants were present in all four population groups at differing allele frequencies ( Table 4 ) .', 'For some loci/populations , the reference genome sequence is not the major allele ( e . g . , SCA17 ) .', 'The genetic consequences of the iMS variants include both sequences that are expected to increase mutability , and sequences expected to decrease mutability .', 'For example , the HOXD13 variant iMS allele is expected to have lower mutability than the reference genome iMS due to the presence of a third interruption that decreases the perfect tandem ( GCG ) 5 repeat to a length below the mutability threshold ( four units for trinucleotide repeats [25] ) .', 'The frequency of this triply-interrupted allele varies from 0 . 76 in the African population to 0 . 26 in European and American populations .', 'The AIB1 locus contains four alternative iMS alleles present at varying frequencies among the populations , one of which is a doubly interrupted allele , leading to greater stabilization of the repeat due to disruption of the ( CAG ) 6 array .', 'For three loci ( DRPLA , SCA1 , and FOXL2 ) , we observed instances of population-specific iMS alleles .', 'DRPLA contained variant alleles in only two of the four population groups studied ( African and American ) , both of which decrease the number of interrupting bases , relative to the reference genome , potentially increasing mutability of the repeat .', 'Finally , we noted an increased number of interruptions within polyglutamine repeats compared to polyalanine repeats , consistent with previous observations about the high propensity of polyglutamine repeats to acquire length and nucleotide polymorphisms [39] .', 'Low indel mutation rates of iMSs ( Figure', '1 ) also are expected to be reflected in their low indel polymorphism levels .', 'To test this , we investigated the levels of heterozygosity and the presence of linkage disequilibrium ( LD ) between interrupted microsatellite alleles caused by indels and neighboring , population-matched SNPs from the 1000 Genomes Phase-1 data .', 'Approximately 30–40% of iMSs display low levels of heterozygosity ( below 0 . 2; Figures S3A–D ) .', 'In fact , we observed a skew towards lower heterozygosity for iMSs as compared to that for perfect microsatellites ( p\u200a=\u200a0 . 028 for Asians; p\u200a=\u200a0 . 066 , p\u200a=\u200a0 . 057 , and p\u200a=\u200a0 . 072 for Africans , Americans , and Europeans , respectively; Kolmogorov-Smirnov test ) .', 'In each of the four populations studied , 4 , 400 to 5 , 000 interruption-causing alleles ( 36–49% of the alleles investigated ) were found to be in moderate LD ( R2>0 . 80 ) with SNPs ( Figure S4 , Table S5 ) , and 686 to 990 alleles ( 6–10% ) were in perfect LD ( R2\u200a=\u200a1 ) with SNPs .', 'Interestingly , certain interruption alleles displayed perfect LD in some , but not all , populations ( Table S5 ) .', 'Generally , iMS alleles in the African population displayed lower levels of LD compared to the other three populations ( Figure S3 ) , likely due to the abundance of low-frequency variants in Africans compared to non-African populations [36] .', 'The exonic iMSs in perfect LD with neighboring SNPs were examined in more detail .', 'Within each population , 6 to 11 of such alleles were identified ( Table S6 ) .', 'For each allele , we examined the phenotype and disease relationships of the linked SNPs using SNPnexus web browser [40]–[42] , and found associations with cancer , neurological , immune , cardiovascular , and metabolic disorders ( Table S6 ) .', 'These associations reiterate a potential for iMSs to modulate disease risk in a population-specific manner .', 'We sought to directly verify the quantitative effect on mutability of a single base substitution interruption within an exonic microsatellite encoded within a human disease gene .', 'We chose the well-established biological model of a population-specific iMS encoded within the APC tumor suppressor gene .', 'In 6% of the Ashkenazi Jewish population , a centrally located iMS ( AAATAAAA ) within an exon of the APC gene is present in the germline as a perfect A8 microsatellite ( AAAAAAAA ) ; this nonsynonymous SNP leads to an I1307K variant , but has no effect on APC protein function [19] .', 'Nevertheless , this population has a greater chance of producing an inactive APC gene in somatic tissues , which increases the risk of colorectal cancer [43] .', 'The proposed mechanism accounting for this observation is the enhanced somatic mutability of the perfect A8 sequence , relative to the interrupted sequence [19] , [44] .', 'We modeled the germline sequences of the perfect and interrupted APC microsatellites , and measured DNA polymerase strand slippage error rates using our established in vitro assay .', 'Briefly , in this analysis , defined tandem repeat sequences are inserted in-frame within a reporter gene .', 'Vectors containing these reporter cassettes are used as templates for in vitro DNA synthesis reactions , and DNA polymerase errors that result in gene inactivation ( frameshift , nonsense or missense mutations ) are scored by genetic selection in E . coli [45] , [46] .', 'To determine the specificity of polymerase errors , independent mutants are isolated , and the DNA sequence changes within the reporter region are determined by dideoxy DNA sequence analysis of purified vector DNA [47] .', 'For these experiments , we examined three DNA polymerases , representing distinct polymerase families and postulated to be required for distinct genome maintenance functions: Pol α , DNA replication; Pol β , DNA repair; and Pol η , translesion synthesis .', 'The accuracy of each polymerase was measured on four DNA templates , representing the complementary strands of the perfect ( A8 and T8 ) and iMS ( A3TA4 and T3AT4 ) alleles in APC ( Figure 3A ) .', 'For the perfect allele templates , the polymerases created +1 A/T insertions , −1 A/T deletions , and A:T to T:A tranversions that lead to TAA nonsense codons ( data not shown ) , which also are the types of inactivating APC somatic mutations observed within tumors from I1307K carriers [44] .', 'For the iMS allele templates , the polymerase indel error frequency was five- to 50-fold lower than that for the perfect allele , depending on the polymerase , demonstrating strand slippage stabilization by this single interruption ( Figure 3A; Table S7 ) .', 'We observed that the interrupting base is rarely removed by these polymerases; the predominant errors ( >95% ) are indels within the remaining perfect tandem repeat tracts ( Figure 3B ) .', 'The frequency of deleting the interrupting base to create a perfect allele was very low ( 9 . 2×10−6 and 2 . 2×10−5 for Pol α and Pol β , respectively ) , relative to other types of polymerase errors ( Table S7 ) .', 'Moreover , the polymerase error frequencies at the residual repeats within the iMS alleles were similar to the error frequencies at similar short tandem repeats located elsewhere within the HSV-tk gene coding sequence ( data not shown ) .', 'These analyses strongly suggest that the single nucleotide interruption within the APC gene leads to the mutational death of the microsatellite .', 'DNA sequence analyses of Pol η errors produced on the interrupted templates emphasized the novel mutational signature of this enzyme within this specific microsatellite motif ( Figure 3C ) .', 'Intriguingly , Pol η has the unique ability to litter this iMS with additional errors , often creating a DNA synthesis product that is more random in sequence than the starting iMS template sequence .', 'Despite this ability , the original interrupting base is maintained in the majority ( 79% ) of Pol η synthesis products .', 'Despite the clear biological significance of iMSs on human genome stability and disease risk , very little is known regarding the biochemical pathways by which interruptions arise in microsatellites .', 'Mutational events to create interrupted alleles could be produced during several cellular mutagenesis pathways , including cytosine deamination events , the creation of abasic sites , endogenous DNA damage-induced mutations and DNA polymerase errors , among others .', 'We used two complementary approaches to gain insight into the potential pathways underlying the production of iMS in the human genome .', 'First , the abundance of polymorphic interruptions and the short evolutionary time since divergence of the four 1000 Genomes population groups allowed us to examine the types of mutations leading to population-specific microsatellite interruptions in detail .', '( We observed a high degree of interruption gain/loss event saturation along primate phylogenetic branches , precluding us from deciphering interruption pathways in this data set .', 'For instance , the resulting numbers of interruptions along the human or chimpanzee lineages since their ∼6 MY split were similar to that along the orangutan lineage since its ∼12 MY split from the human lineage ( Figure S5 ) ) .', 'Second , the fact that DNA polymerases can create interruption errors during in vitro synthesis of microsatellite-containing templates [45] , [46] , [48] afforded us the opportunity to examine one biochemical pathway- namely , polymerase errors during DNA synthesis .'], ['Early studies of microsatellite interruptions demonstrated reduced mutation rates at a few iMS loci , as compared with perfect alleles of the same repeat number [28]–[30] , [44] .', 'A higher mutability of microsatellites was observed for interruptions closest to the repeat tract ends , as compared with centrally located interruptions [31] , [33] , [53] , [54] .', 'Such studies suggested that interruptions might effectively divide microsatellites into shorter repeat runs .', 'Within the interrupted repeat itself , the mutation rates of the individual arms depend on the lengths of perfect tracts remaining within the iMS allele [55] .', 'Here , we provide a detailed , genome-wide analysis of the mutability of perfect and interrupted MSs in completely sequenced primate genomes .', 'For the four motif sizes examined , interruptions significantly reduced mutability when present', '( a ) within shorter microsatellites ,', '( b ) in multiple numbers ( i . e . , two interruptions per microsatellite ) , or', '( c ) near the center of the microsatellite ( Figure 1 ) – all of which give rise to a shorter perfect repeat tract .', 'Importantly , the magnitude of the effect of interruptions on microsatellite allele length variation ranged from a few-fold to several orders of magnitude for loci across the genome .', 'We also report here that the perfect microsatellites in the human reference genome analyzed here ( ≤10 units in length ) are frequently found as iMS polymorphisms within the genomes of individuals from four population groups .', 'Although the majority of iMS alleles were shared among all groups , many of the iMS alleles we detected were specific to only one population group , or shared between subsets of population groups ( Figure 2 ) .', 'Our quantitative results for the stabilizing effects of interruptions in short microsatellites are biologically relevant here , as the vast majority of iMSs we identified in human genomes are within short microsatellites , just above the length threshold .', 'Therefore , interruptions are expected to have a strong effect on stabilizing such microsatellites .', 'Thus , iMSs are a likely source of population-specific genetic variants that can affect the stability of individual genomes by reducing the mutability of microsatellites .', 'To the best of our knowledge , this is the first report of iMSs as an abundant source of population-specific genetic modifiers in the human genome .', 'The full abundance of iMSs within the human genome must await future studies , when improvements in sequencing technology read length and accuracy will allow the interrogation of all microsatellite motif sizes , lengths , and sequences that are present within individual genomes .', 'The APC tumor suppressor gene illustrates a provocative example in which a single , population-specific , germline SNP can affect disease risk by altering the mutagenic potential of a microsatellite sequence .', 'Our data directly support the previous model that the perfect [A8/T8] allele creates a hypermutable region within the APC gene , leading to cancer predisposition [19] .', 'We measured DNA polymerase strand slippage error rates that are up to 50-fold lower during replication of the iMS sequences [A3TA4/T3AT4] , compared to the perfect sequences [A8/T8] ( Figure 3 ) .', 'Previous biochemical studies of trinucleotide microsatellites have shown that interruptions decrease slipped strand formation [56] and decrease the thermostability of secondary structures formed by repetitive sequences [57] .', 'Our results advance these studies by demonstrating that the mechanism of reduced mutability by an interruption within a mononucleotide A/T allele is lowered polymerase strand slippage errors during DNA synthesis .', 'Expanding on the APC gene observation that SNPs can create perfect microsatellites and hypermutable sequences in disease states , we identified ∼3 , 000–4 , 000 genes ( depending on the population group ) that are perfect in the reference genome , but contain iMS within exonic regions ( Figure 2 ) .', 'The exonic iMS alleles that are specific to only one or two populations likely represent a pool of genes that are at a risk for increased mutation in the other population groups .', 'Madsen and colleagues reported that short tandem repeats/microsatellites in exons are overrepresented among human genes associated with cancer and immune system diseases [58] .', 'We observed that while European-specific iMSs are enriched in genes associated with immunological function , African-specific iMSs are enriched in genes associated with neurological function .', 'Thus , population-specific differences in microsatellite architecture ( perfect versus interrupted ) may be a widespread mechanism by which genetic ancestry impacts individual disease risk .', 'While our focus has been on comparing population groups , our FST analysis indicated that many iMS alleles are not fixed within population groups , thus potentially providing a rich source of individual genetic variability .', 'Perfect microsatellites are at a higher risk for microsatellite expansion mutations that are causative for numerous neurological/neurodegenerative diseases [3] , and the presence of interrupted alleles has been well documented to decrease disease risk .', 'We investigated several genes previously described as harboring disease-associated , coding iMS alleles [38] .', 'The genetic consequences of the iMS variants we identified include both sequences that are expected to increase mutability , and sequences that are expected to decrease mutability .', 'Various AIB1 iMS alleles have been noted previously in a survey of European DNA samples [59] , consistent with the allelic distribution we observed for the 1000 Genomes European population group .', 'One of the iMS variants we identified within AIB1 occurs at a much higher allele frequency in the African population , and is expected to display higher mutability than the reference sequence , due to an increased perfect tandem repeat tract length .', 'The two HOXD13 iMS alleles we identified were observed previously in a pedigree analysis of 16 synpolydactyly families [60] .', 'Importantly , repeat expansions in these families segregated with the disease phenotype; however , the iMSs were retained in all of the expanded alleles .', 'Recently , amyotophic lateral sclerosis patients have been described as having moderately expanded SCA2 iMS alleles that retain at least one of the interruptions [20] , [61] .', 'Both microsatellite length and purity ( interruption ) SCA1 and SCA2 polymorphisms have been described among unaffected individuals [62] , [63] , consistent with the iMS variant alleles we detected in this study .', 'The pathways by which iMSs arise in genomes have not been extensively studied .', 'Several cellular mechanisms could account for the production of iMS alleles in genomes , including ( but not limited to ) endogenous DNA damage-induced mutations and DNA synthesis errors during DNA replication , repair and/or recombination .', 'The types of iMS ultimately observed in human genomes will be further shaped by DNA repair pathways and selection , which will serve to reduce the number of and narrow the types of mutational events within microsatellites .', 'We demonstrate here that base substitutions are the primary type of iMS present in individual human genomes .', 'We also used our established biochemical assay to determine the potential contribution of errors created by three distinct DNA polymerase families to the formation of iMS alleles .', 'For the microsatellite templates and types of detectable errors examined , we observed that genome stabilizing microsatellite interruptions are created most frequently in vitro by error-prone , specialized Pols η and κ , while replicative Pols α and δ rarely created interruptions ( Figure 5 ) .', 'The generality of our observations for all microsatellite sequences and human polymerases is not known , and must await future experimental analyses .', 'Nevertheless , we observed that DNA Pol η is very efficient at making interruptions within perfect microsatellites and creates multiple errors within a single DNA synthetic event .', 'Pol η also creates base substitution errors within the tandem repeat tracts of iMS templates , with the net result being a more random sequence .', 'DNA Pol η serves several important functions in human genome stability .', 'Germline mutations leading to loss of Pol η activity causes the cancer predisposition syndrome , xeroderma pigmentosum-variant [64] , and enhanced cellular UV sensitivity [65] .', 'Pol η has been well- characterized biochemically , and is capable of accurate translesion synthesis across UV photoproducts and other DNA lesions [64] , [66] .', 'Huma
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation .', 'However , human polymorphism levels at interrupted microsatellites ( iMSs ) are not known at a genome-wide scale , and the pathways for gaining interruptions are poorly understood .', 'Using the 1000 Genomes Phase-1 variant call set , we interrogated mono- , di- , tri- , and tetranucleotide repeats up to 10 units in length .', 'We detected ∼26 , 000–40 , 000 iMSs within each of four human population groups ( African , European , East Asian , and American ) .', 'We identified population-specific iMSs within exonic regions , and discovered that known disease-associated iMSs contain alleles present at differing frequencies among the populations .', 'By analyzing longer microsatellites in primate genomes , we demonstrate that single interruptions result in a genome-wide average two- to six-fold reduction in microsatellite mutability , as compared with perfect microsatellites .', 'Centrally located interruptions lowered mutability dramatically , by two to three orders of magnitude .', 'Using a biochemical approach , we tested directly whether the mutability of a specific iMS is lower because of decreased DNA polymerase strand slippage errors .', 'Modeling the adenomatous polyposis coli tumor suppressor gene sequence , we observed that a single base substitution interruption reduced strand slippage error rates five- to 50-fold , relative to a perfect repeat , during synthesis by DNA polymerases α , β , or η .', 'Computationally , we demonstrate that iMSs arise primarily by base substitution mutations within individual human genomes .', 'Our biochemical survey of human DNA polymerase α , β , δ , κ , and η error rates within certain microsatellites suggests that interruptions are created most frequently by low fidelity polymerases .', 'Our combined computational and biochemical results demonstrate that iMSs are abundant in human genomes and are sources of population-specific genetic variation that may affect genome stability .', 'The genome-wide identification of iMSs in human populations presented here has important implications for current models describing the impact of microsatellite polymorphisms on gene expression .']
['Microsatellites are short tandem repeat DNA sequences located throughout the human genome that display a high degree of inter-individual variation .', 'This characteristic makes microsatellites an attractive tool for population genetics and forensics research .', 'Some microsatellites affect gene expression , and mutations within such microsatellites can cause disease .', 'Interruption mutations disrupt the perfect repeated array and are frequently associated with altered disease risk , but they have not been thoroughly studied in human genomes .', 'We identified interrupted mono- , di- , tri- and tetranucleotide MSs ( iMS ) within individual genomes from African , European , Asian and American population groups .', 'We show that many iMSs , including some within disease-associated genes , are unique to a single population group .', 'By measuring the conservation of microsatellites between human and chimpanzee genomes , we demonstrate that interruptions decrease the probability of microsatellite mutations throughout the genome .', 'We demonstrate that iMSs arise in the human genome by single base changes within the DNA , and provide biochemical data suggesting that these stabilizing changes may be created by error-prone DNA polymerases .', 'Our genome-wide study supports the model in which iMSs act to stabilize individual genomes , and suggests that population-specific differences in microsatellite architecture may be an avenue by which genetic ancestry impacts individual disease risk .']
['sequencing techniques', 'medicine and health sciences', 'genetic dominance', 'population genetics', 'cancer risk factors', 'oncology', 'genome sequencing', 'mutation', 'dna replication', 'dna', 'molecular biology techniques', 'autosomal dominant diseases', 'huntington disease', 'dna synthesis', 'genome complexity', 'genetic polymorphism', 'autosomal dominant traits', 'personalized medicine', 'comparative genomics', 'molecular biology', 'clinical genetics', 'biochemistry', 'genetic causes of cancer', 'nucleic acids', 'genetics', 'biology and life sciences', 'genomics', 'evolutionary biology', 'computational biology', 'human genetics']