Tri-allelic pattern of short tandem repeats identifies the murdereramong identical twins and suggests an embryonic mutational origin

Li-Feng Wang a,d,1,*, Ying Yang a,b,d,1, Xiao-Nan Zhang a,b,d, Xiao-Liang Quan c,Yuan-Ming Wu a,b,d,**aDepartment of Biochemistry and Molecular Biology, Xi’an, Shaanxi, ChinabCenter for DNA Typing, Xi’an, Shaanxi, Chinac Public Security Bureau, Qi’shan, Shaanxi, Chinad Fourth Military Medical University, Xi’an, Shaanxi, China

A R T I C L E I N F O

Article history:Received 8 October 2014Received in revised form 13 January 2015Accepted 29 January 2015

Keywords:Tri-allelic patternSTR mutationIdentical twins

A B S T R A C T

Monozygotic twins can be co-identified by genotyping of short tandem repeats (STRs); however, fordistinguishing them, STR genotyping is ineffective, especially in the case of murder. Here, a rarelyoccurring tri-allelic pattern in the vWA locus (16, 18, 19) was identified only in the DNA of one identicaltwin, which could help to exonerate the innocent twin in a murder charge. This mutationwas defined asprimary through genotyping of the family and could be detected in blood, buccal and semen samplesfrom the individual; however, two alternative allele-balanced di-allelic patterns (16, 18 or 16, 19) weredetected in hair root sheath cells. Such a kind of segregation indicates a one-step mutation occurs in cellmitosis, which is after embryonic zygote formation and during the early development of the individualafter the division of the blastocyte. Sequencing revealed the insertion between the allele 18 and 19 is arepeat unit of TAGA/TCTA (plus/minus strand), which belongs to “AGAT/ATCT”-based core repeatsidentified from all tri-allelic pattern reports recorded in the STR base and a detailed model was proposedfor STR repeat length variation caused by false priming during DNA synthesis. Our model illustrates thepossible origination of allele-balanced and unbalanced tri-allelic pattern, clarifies that the genotypes ofparent–child mismatches, aberrant di-allelic patterns, and type 1 or 2 tri-allelic patterns should beconsidered as independent, but interconnected forms of STR mutation.

ã 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

STR (short tandem repeat) loci consist of short, repetitivesequence elements throughout the genomes of a wide range ofspecies [1,2]. Alleles of STR loci are differentiated by the number ofcopies of the repeat sequence contained within the amplifiedregion. For its variety and stability, STRs arewidely used for geneticfingerprinting [3,4]. In the field of forensics, STR screening providesa high degree of error-free data on personal genetic identity [5,6],

while being robust enough to survive degradation under non-idealconditions. Profiles of certain STR sets are potent and can be used todetermine whether twins are monozygotic or dizygotic [7].However, it is nearly impossible to discriminate betweenmonozygotic twins, especially in the case of rape or paternitytesting, which creates a limitation in forensic testing and begs thequestion: Could identical twins pull off the perfect crime and avoidconviction?

A missing girl was discovered raped and murdered in QishanCountry, Shaanxi, China. The semen detected at the crime scenewas attributed to one of her neighbors; however, his identical twinbrother, who lived with the suspect, needed to be ruled out. Nofingerprint or further biological evidence was available. Addition-ally, no commercial protocol was available to forensicallydistinguish identical twins by DNA testing. Under such circum-stances, newmethodology was needed to distinguish between themonozygotic twins.

* Corresponding author at: Fourth Military Medical University, Department ofBiochemistry and Molecular Biology, Xi’an, China. Tel.: +86 029 87971127.** Corresponding author at: Fourth Military Medical University, Center for DNATyping, Xi'an, Shaanxi, China. Tel.: +86 029 84774978.

E-mail addresses: lfwang@fmmu.edu.cn (L.-F. Wang), wuym@fmmu.edu.cn(Y.-M. Wu).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fsigen.2015.01.0101872-4973/ã 2015 Elsevier Ireland Ltd. All rights reserved.

Forensic Science International: Genetics 16 (2015) 239–245

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2. Materials and methods

2.1. Subjects

Our study was approved by the ethical review board of theFourth Military Medical University (FMMU). All participantsprovided written consented for the use of their DNA typing datafor research purposes. Semen sample is collected from the crimescene. Then blood sample was took from both of the identicaltwins, their parents, and their younger sister; additional buccal,semen and hair samples are collected from the criminal candidate2.

2.2. DNA extraction

Corresponding peripheral blood from selected individuals wastreated with EDTA-Na2 for anticoagulation. Genomic DNA (germ-line source)was extracted fromblood by the RelaxGene Blood DNASystem DP319-02 (TIANGEN, Beijing, China). The concentration ofgenomic DNA was measured with the NanoDrop 2000 (ThermoScientific, Wilmington, DE, USA). For further analysis, the sampleswere diluted to defined concentrations. DNA from semen, buccalmucosa and hair follicles were extracted by chelex-100 method.The filter paper spottingwith semen, the swab stickingwith buccal

mucosa and single hair follicle were put into the eppendorf tubeseparately, then 200ml of 5% chelex-100 (Bio Rad Laboratories,Hercules, California, USA) and 4ml proteinase k (Tiangen, Beijing,China) were added, the tubes were incubated in a water bath for60min at 56 �C followed by a 10min incubation at 99 �C. Aftercooling, the samples were vortexed briefly and centrifuged. Thesupernatant was used as a substrate in PCR reactions.

2.3. STR typing

For the amplification of short tandem repeats, the commercialAmpFlSTR SinofilerTM kit (Applied Biosystems, Foster City, CA,USA) was used according to the manufacturer’s instructions withthe ABI GeneAmp PCR system 9700. When considered necessary,the DNA concentration and number of cycles were modified forPCR optimization. PCR fragments were separated by capillaryelectrophoresis (using POP-4 polymer) in an ABI PRISM310GeneticAnalyzer (Applied Biosystems, Foster City, CA, USA).

2.4. Primer design and gene sequencing

Based on the human vWA gene sequence, a pair of primers(sense: 50 GAT ACA TAG GAT AGA TAG AGA CAG G30; antisense:50ggt aga gtt ccc acc ttc30) was designed to amplify the vWAsequence from genomic samples. PCR conditions used were: 3min

[(Fig._1)TD$FIG]

Fig.1. Three allele bands at the vWA locus rule out C2 as the perpetrator.16 STR loci profiles of blood samples from the identical twins, criminal candidate 1 (C1) and criminalcandidate 2 (C2) are shown. (A) Blood sample profile of C1; (B) blood sample profile of C2; (C) the allelic pattern at the vWA locus is magnified and comparedwith the patternof the semen detected at the crime scene; (D) vWA profile ladder.

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at 95 �C, and then 10 cycles of 95 �C for 30 s, 56 �C for 30 s and 72 �Cfor 30 s, with a decrease of the annealing temperature by 0.5 �C percycle. Subsequently, 20 further cycles at the condition of the lastcycling step (51 �C annealing temperature) were performedfollowed by incubation for 7min at 72 �C. PCR products wererun on 1.5% agarose gels and visualized by ethidium bromidestaining. DNA bands of the expected length rangewere gel purifiedusing the TIANgel midi purification kit (Tiangen biotech, Beijing,China) and subcloned into the pGM-T vector (Tiangen biotech,Beijing, China). PCR products were analysed on anABI310 sequencing system using T7 primers and the Big dye cyclesequencing kit (Applied Biosystems, Foster City, CA, USA).

3. Result

3.1. Three allele bands discriminate perpetrator from identical twins

Blood samples from the identical twins were collected forstandard forensic genotyping by the AmpFlSTR SinofilerTM kit. The16 STR loci designed for Asians were tested. The profiles of thetwins matched perfectly except for the presence of three allelebands at the vWA locus for criminal candidate 2 (C2) as comparedto a di-allelic pattern for criminal candidate 1 (C1) (Fig. 1). Theprofile of the vWA locus in semen from the crime scene matchedthe C1 blood sample, indicating that this unique mutation can ruleout C2 as the perpetrator.

3.2. Onset and penetration of tri-allelic patterns in the twins’ family

Because tri-allelic patterns are seldom identified, even inworldwide populations, we sought to investigate the onset andpenetration of this pattern in the twins’ family. Blood samples fromthe father, mother and sister were collected for DNA typing. Other

than C2, no further tri-allelic patterns was found (Fig. 2A–E).Semen and buccal samples verified the characteristic tri-allelicpattern of the blood sample fromC2, inwhich the area of themajorallele (16 in Fig. 2D, F andG) is nearly always equal to the sumof theareas of the other two minor alleles (18 and 19 in Fig. 2D, F and G).To test whether the tri-allelic pattern may have arisen fromaneuploidy, root sheath cells of anagen head hairs were collectedfrom 4 individual hairs of C2. Interestingly, the tri-allelic pattern atthe vWA locuswas not observed in any picked individual hairs, andinstead, an allele-balanced di-allelic 16, 18 (Fig. 2H1) or 16, 19(Fig. 2H2–H4) pattern could be clearly detected in each hair, whichindicates that the vWA locus allele type 18 may have mutated into19, and aneuploidy or the presence of three alleles cannot explainthe tri-allelic pattern in this case. The allelic pattern differencebetweenmonozygotic twins, even in different tissue samples fromthe same individual, imply that the tri-allelic pattern probablyarose after the division of the blastocyte (after monozygous zygoteformation, one blastocyte keeps unaffected, but the othergradually gains the STR mutation) and before the formation ofthe respective germ layer (tridermogenesis) in mitosis duringembryonic development (so each germ layer from the mutationevent affected individual blastocyte obtain a mixture of unaffectedand mutant di-allelic patterns).

3.3. Insertion in tri-allelic patterns identified through directsequencing

As a tetranucleotide microsatellite, the vWA STR consists ofTAGA repeatswith CAGA inserts (gDNA plus strand), which are alsopresent in GenBank as TCTA with TCTG inserts (view as minusstrand). Sequencing was performed on the vWA STR loci from theindividual hair samples of C2 to provide some insight into how themutation might have arisen. As shown in Fig. S1, the only

[(Fig._2)TD$FIG]

Fig. 2. Evidence for the onset and penetration of the tri-allelic pattern based on genotyping of C2 familymembers and different C2 sample sources. Allelic patterns at the vWAlocus are shown for blood samples from the twins’ father (A), the twin’s mother (B), the C1 twin (C), the C2 twin (D) and the twins’ sister (E). Additionally, the patterns ofbuccal and semen samples from C2 are shown in F and G; and the individual patterns of 4 hairs from C2 are shown in H1–H4.

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difference between alleles 18 and 19 is in the number of TAGArepeat unit.

3.4. Model for the origin of insertion

How could a TAGA insertion occur? A mechanism of replicationslippage is detailed in our model (Fig. 3). During eukaryotic DNAreplication, the origin sites and the 50 ends of the Okazaki fragmentare initially shielded by RNA primers, which are then removed bythe exonuclease MF1. Unlike DNA polymerase I (pol I) inprokaryotes, which specifically seals the nicks between newlysynthesized fragments, in eukaryotes a switch occurs after theinitiation of replication from DNA pol a to DNA pol d/e. During theswitch, the 30 end is unwound and assembled as primer onto pold/e, which coordinates the functions of DNA helicase, DNAsynthesis and excision repair. The process of primer assemblymight be especially prone to error for repeated sequences, such asthe TAGA/TCTA (plus/minus strand) of the vWA loci in our case.

When priming, the sequence could form a mini-hairpin whilebinding with the template sequence in a nearly perfect base pairmatch. In such case, the expansion of one repeat unit would occuron the primer-containing strand, but not the parental strand.Althoughmost of the loop-based DNAmismatch during replicationwould be corrected by DNA repair, a small non-repaired fractionmay have been randomly left in the short DNA synthesis interval inearly embryonic development (from blastocyte to the formation ofgerm layer), thus giving rise to a mixture state of STR mutation. Infact, mutations related to DNA mismatch repair genes (MLH1,MSH2, MSH6, PSM2, MUTYH) have been reported as the motivatingforce of colorectal cancer syndrome characterized by pronouncedmicrosatellite instability (MSI) [8].

Our model is not limited to a one unit-based primer/templateloop, but could also explain the formation of two, even three ormore unit-based loops (Fig. 3). This means that our false primingreplication slippage model no longer has a one-repeat unitlimitation for single-step mutation, as proposed [9]. However,

[(Fig._3)TD$FIG]

Fig. 3. Proposed mechanism of false loop priming to produce STR repeat length variation. During the process of DNA replication, pol d/e is loaded at the end of the alreadysynthesised Okazaki fragments to fill the gap remaining after the degradation of the RNA primers. The 30 ends of the DNA fragment are unwound and assembled as primeronto pol d/e. We propose that the vWA loci repeat sequence of TAGA could form a loop based mini-hairpin, but also retain its ability to bind with the corresponding templatesequence in a nearly perfect base pair match. The base view of the local structure shows that a primer loop with one repeat unit would result in the expansion of one repeatunit on the primer-containing strand. Structures that could produce two or three repeat units are also indicated.

Table 1Basics of four typical aberrant STR patterns in genotyping.

Aberrant STRpatterns

Parent–childmismatch

Aberrant di-allelic patterns Type 1 tri-allelicpatterns

Type 2 tri-allelic patterns

Changed band A new bandsubstitution

No new band An additionalband

An additional band

Di-allelic balance Balance Imbalance Balance ImbalanceAllele number Two Two or more Two Two or more?Possiblemechanisms

Replicationslippage

Regional STR loci duplication; replicationslippage or aneuploidy

Replicationslippage

Regional STR loci duplication followed by replicationslippage or aneuploidy?

Onset time Meiosis Mitosis or meiosis Mitosis Mitosis or meiosisHeredity New band Patterns Partial new band Patterns

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the longer the loop stem, the easier it would be to be detected andrepaired. As amatter of fact, 89.8% of the knownmutational eventsappeared to involve the loss or gain of a single repeat unit [10].

3.5. Repeat unit biases in tri-allelic patterns and parent-childmismatches

A total of 326 tri-allelic pattern reports have been recorded inthe STR base (Web Resources 1), which have been localized to34 STR loci among the 41 loci within the human genome, includingchromosome Y (Table S1). Structurally speaking, 22 of them,according to 203 (62.27%) of reports, have “AGAT/ATCT” (50 to 30,plus strand/minus strand) – based core repeat units, whichcomprise 5 different tetranucleotide repeats (TAGA/TCTA, AGAT,GATA/TATC) and 1 pentanucleotide repeat (AGAGAT). Three otherkinds of “AT or GC”-based tetra nucleotide repeats (AATG, ATTT,and TGCC) in 4 STR loci are the basis for 27 tri-allelic reports(8.28%). These repeats, which comprise 70.55% of all tri-allelicpattern reports combined, may share a common mechanism offalse loop priming as shown in Fig. S2. The remaining 96 (29.45%)of the reports of 8 STR loci can be grouped into 5 “AG or TC”-basedtetra- (AAAG, AAGG, AGAA, GAAA,GGAA) and 2 penta- (AAAGA,TTTTC) nucleotides, and cannot be considered in the false looppriming model.

Data from parent–child mismatches also have shown certaincore repeat unit biases. Among 33 instances ofmismatch in a studyof 27,086 parent–child pairs from Brazil [11], 26 (78.79%) have“GATA” repeat units (belong to the D5S2501, D10S1237, D15S657,and D18S1270 loci) which are “AGAT/ATCT”-based core repeats;and the other 7 (21.21%) have “AG”-based (4 AAGG and 3 GGAA)repeats in the D1S1612 and D4S2431 loci. The composition of theSTR repeat unit could decide the structure of the mini-hairpin formismatch priming.

3.6. Basic properties of aberrant STR

The basic properties of four possible aberrant STR patterns aresummarized in Table 1. The STR sequencing data from themonozygotic twins in this study supports the mechanism of type1 tri-allelic pattern origination, which is non-heritable (Fig. 4). As aone-step slippage mutation, both wild type and mutant STRgenotypes are maintained in different descendant cell masses inthe individual following DNA replication, although only onegenotype is observed in a single cell, as reflected by the analysisof the anagen head hairs. Accordingly, the amplification productsof the blood, semen and buccal samples equaled the summedproducts of the mutation event that involved alleles that werediploid-balanced on a single cell basis (Fig. 4A).

4. Discussion

STR screening is never thought to be used for discriminationone from the other between the monozygotic twins, even if theyinvolved in criminal case. Model technology such as copy-number-variation profiles [12], DNA methylation profiles [13], and nextgeneration sequencing [14] are all potential for a solution;however, just before any of those techniques to be adopted intothis case, the real criminal was “unluckily” identified through thetraditional STR test by the accidental emerge of a tri-allelic pattern.Then, how could it happen?

STRs could also be called microsatellites according the size ofthe repeated unit (up to 9nt). They are particularly interesting froman evolutionary point because they are unstable, mutating athigher frequencies than other sequences in the genome by themechanism of slippage mutations [15]. Typically, slippagemutations in repetitive DNA occur about once per 1000 gener-ations, highlighted by the fact that DNA polymerases produce

[(Fig._4)TD$FIG]

Fig. 4. Diploid balanced STR tri-allelic patterns are induced by a mitotic one-step somatic mutation. If a tissue sample is developed from a single cell (A), and the mutationoccurs during the first round of mitosis, then 50% of the daughter cells (A1) will be comprised of the mutant allele (19) and 50% will be comprised of the wild type allele (18).Both populations of cells would have an equal amount of the unaffected allele (16) and the affected allele (18/19) to produce a 2:1:1 type of tri-allelic pattern (X). However, ifthere already have 2 cells (a +b) and the mutation occur in only one of them (A1), a 4:3:1 type of tri-allelic pattern would be observed (Y).

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errors within microsatellites at a 10- to 100-fold higher frequencythan that of frameshifts in coding sequences [16]. While it is truethat STRs that mainly occur in regions of low gene density could beconsidered as nonfunctional ‘junk’ DNA, the recent availability ofwhole genome sequences has revealed that microsatellites are alsopresent in coding regions, promoters and other regulatory regions[17]. Through the variation of just a few repeat units, flies can resettheir internal molecular clocks to changes in their environments[18]. The remarkable variation in dogmorphology can be explainedthrough the functional variation of a repeat-containing develop-mental gene [19]. Moreover, insertion of an interspersed elementfrom retrotransposons might also be favored at sites with a pre-existing microsatellite [20].

Types of aberrant STR presented in population studiesmay helpus know more about its characters and potential origin. Besidethose analyzed in our works, abnormal di-allelic patterns can alsooccur in cases in which a mutated allele is incidentally changedinto the same STR type of the unaffected allele (Fig. S3 A1). Theaffected individual would then acquire a diploid-imbalanced di-allelic STR typewith higher levels of the unaffected allele. Based onour newfindings, we further propose an originationmodel for type2 diploid-imbalanced tri-allelic patterns, in which the amount ofthe three STR allele types is equal (Fig. S3 B1). The formation of thetype 2 tri-allelic pattern could arise from a multi-step somaticmutation event, which involves at least one step of regional STRloci duplication, in combination with a replication slippage eventin which one of the STR loci is duplicated. Such sequential eventscould happen across more than one generation and could takeplace either in meiosis or in mitosis, resulting either in equalamounts of the unaffected and the mutant STR loci (Fig. S3 B1), oran imbalanced di-allelic pattern (Fig. S3 C1 and C2), depending onwhich STRs are affected. If a multi-step mutation event happensbefore or during germ layer formation, a somatic mutation couldbe transferred into the germline, and solely under such acircumstance would this type 2 tri-allelic pattern be heritable[21,22].

If loop-based mismatch priming had occurred before meiosis, adi-allelic mismatch rather than the tri-allelic pattern would beexpected [11]. For mutations that developed before meiosis, eachgerm cell could only contain a single allele type to form a normaldi-allelic STR genotyping pattern (Fig. S4). From this perspective,we can consider parent–child mismatch and tri-allelic patterns asdistinct genetic processes, even if they may share the samemutational mechanism of replication slippage (Table 1). Replica-tion slippage caused by mismatch between DNA strands beforemeiosis is a widely accepted mechanism to explain variations inthe lengths of short sequence repeats [23] and is furtherdemonstrated by the 33 mismatches in the study of parent–childpairs in Brazil [11]. However, their clarification of tetranucleotidemicrosatellite mutation primarily occurs in the paternal germina-tive cells is pale for explain how could such a kind of mutation takeon as tri-allelic patterns. Besides, many types of unbalanced di-allelic STRs for which the proportions of minor alleles range from50% to 10% (also defined as aberrant di-allelic patterns [21]) havebeen reported as a consequence of the extensive use of genotypingtechniques in forensic sciences, paternity testing, molecularecology for population or disease, and even molecular taxonomy.The regional STR loci duplication model developed from ourfindings provide amore insight to interpret all those abnormal STRtypes.

STR polymorphisms have been considered as useful functionalelements that facilitate swift evolution and rapid adaptation tochanging environments [17]. Our refined replication slippagemodel for the induction of aberrant STR patterns may provide analternative insight into STR-based genetic diversity, hereditarydisease and even species evolution.

Author’s contribution

Conceived and designed the experiments: Yuan-Ming Wu,Li-Feng Wang. Performed the experiments: Yuan-Ming Wu,Li-Feng Wang, Xiao-Nan Zhang, Ying Yang, Xiao-Liang Quan.Analyzed the data: Li-Feng Wang, Ying Yang. Wrote the paper:Li-Feng Wang

Web resource

http://www.cstl.nist.gov/biotech/strbase/tri_tab.htm06/11/2013.

Acknowledgements

We especially thank Professor Suocai Su for a detailed literatureediting and proofreading the manuscript. Special thanks also go toProfessor Cornelius F. Boerkoel for valuable suggestions inmanuscript construction. This project is supported by NationalNatural Science Foundation of China (No. 30901359) and the KeyProject Foundation of Shaanxi Science & Technology Commission(No. 2012SF2-01-7). The authors have declared that no competinginterests exist.

Appendix A. Supplementary data

Supplementary data associatedwith this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fsigen.2015.01.010.

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