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 Table of Contents  
Year : 2019  |  Volume : 5  |  Issue : 3  |  Page : 168-171

Profile interpretation of extremely long alleles at DYF387S1 and DYS447 migrated into allele range of adjacent loci

1 Zhejiang Key Laboratory of Forensic Science and Technology, Institute of Forensic Science of Zhejiang Provincial Public Security Bureau, Hangzhou, P.R. China
2 Faculty of Forensic Medicine, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, P.R. China

Date of Web Publication18-Sep-2019

Correspondence Address:
Hailun Nan
Faculty of Forensic Medicine, Zhongshan Medical School, Sun Yat-Sen University, 74# Zhongshan Road II, Guangzhou 510089
P.R. China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_10_19

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Two rare cases of long alleles at Y-chromosome short tandem repeat (Y-STR) loci (DYF387S1 and DYS447) were identified when two father–son pairs were analyzed by multiplex amplification. “Null alleles” were observed at DYF387S1 and DYS447, and duplicated alleles were displayed at DYS533 and DYS19. We secondly amplified DYF387S1, DYS533, DYS447, and DYS19 loci by singleplex polymerase chain reaction (PCR) and sequence analysis of the long alleles at DYF387S1 and DYS447 loci. The results showed that alleles from DYF387S1 (allele 55) and DYS447 (allele 41) were longer than their common sizes in the allelic ladder ranges (33–42 for DYF387S1 and 18–30 for DYS447) and located in the neighboring loci (DYS533 and DYS19, respectively). Therefore, to identify these cases involving this unusual phenomenon, not only re-amplification using the same kit but also additional amplification (using alternative multiplex kits with different adjoining markers or additional singleplex PCR amplification) should be performed to avoid misinterpreting Y-STR profiles.

Keywords: DYF387S1, DYS447, long allele, profile interpretation, Y-chromosome short tandem repeat migration

How to cite this article:
Wu WW, Hao H, Lu D, Nan H. Profile interpretation of extremely long alleles at DYF387S1 and DYS447 migrated into allele range of adjacent loci. J Forensic Sci Med 2019;5:168-71

How to cite this URL:
Wu WW, Hao H, Lu D, Nan H. Profile interpretation of extremely long alleles at DYF387S1 and DYS447 migrated into allele range of adjacent loci. J Forensic Sci Med [serial online] 2019 [cited 2022 May 18];5:168-71. Available from: https://www.jfsmonline.com/text.asp?2019/5/3/168/267148

  Introduction Top

The male-specific Y-chromosome short tandem repeat (Y-STR) loci are routinely used in forensic DNA analysis, especially in cases with evidence containing a mixture of male/female material and for patrilineal identification.[1] Nowadays, fluorescently labeled primers for multiplex amplification are widely applied to the analysis of Y-STRs. Polymerase chain reaction (PCR) products are separated by size and dye color using capillary electrophoresis. STR genotyping is performed by comparing the allele sizes in each sample to the sizes of alleles present in an allelic ladder, which contains alleles that are common in the population.[2]

Since STRs consist of a variable number of tandem repeats, very long STR alleles outside the common allelic size range have been observed at some autosomal STR loci. Such long alleles can extend into adjacent loci, leading to incorrect allele designation.[3],[4],[5],[6] However, extremely long alleles at Y-STR loci are very rare. We here report two cases of long alleles occurring at loci DYF387S1 and DYS447.

  Materials and Methods Top

Buccal swab samples from two father–son pairs were collected from Han Chinese in Eastern China. Informed consent was obtained from the participants. DNA from the buccal cells was extracted using the DNA IQ System (Promega Corporation, Madison, WI, USA). The PCR amplification was performed using three Y-STR multiplex PCR reactions: Yfiler ® Plus PCR Amplification Kit (Life Technologies Corporation, Carlsbad, USA), AGCU GFS Y-STR Kit, and AGCU Y24 STR Kit (AGCU ScienTech Inc., Wuxi, Jiangsu, China). First, both Yfiler ® Plus PCR Amplification Kit and AGCU GFS Y-STR Kit were used in our two cases. Then, the genotype of DYS19 was confirmed using the Yfiler ® Plus PCR Amplification Kit. All assays were performed in accordance with the manufacturer's recommendations. PCR was carried out in an ABI PRISM ® GeneAmp ® 9700 PCR System (Applied Biosystems, Foster City, USA). PCR products were separated and detected on an ABI PRISM ® 3500xL Genetic Analyzer (Applied Biosystems) and were analyzed using GeneMapper ® ID v. 1.4 software (Applied Biosystems), following the manufacturer's protocol. Control DNA 007 or 9948 provided in the kits was genotyped as a standard reference.

To confirm the alleles falling outside of the marker size range, additional singleplex PCR was performed to amplify DYF387S1, DYS533, DYS447, and DYS19 loci, and sequencing analyses of the long alleles of DYF387S1 and DYS447 were performed by AGCU ScienTech Inc.

The father–son biological relationship was confirmed by the finding of 38 autosomal STRs with paternity index values above 100,000.

  Results and Discussion Top

Two father–son pairs showed a null allele at the smaller size locus and a duplicated allele at the larger neighboring locus. In the first pair, the rare type of Y-STR was observed by the Yfiler ® Plus PCR Amplification Kit. Thus, at locus DYF387S1, there were no alleles detected within the marker range of this locus. However, the neighboring locus DYS533 carried two alleles labeled as 12 and off-ladder (OL, as defined by GeneMapper™ ID software) [Figure 1]. Similarly, the second pair did not show the Y-allele at locus DYS447 with the AGCU Y24 STR Kit, and two duplicated alleles labeled as 14 and OL were observed at the adjacent locus DYS19 [Figure 2].
Figure 1: The electrophoregram of the father in the first pair using Yfiler® Plus Polymerase Chain Reaction Amplification Kit. The plot shows that no allele peaks are observed at locus DYF387S1, but allele peaks labeled 12 and OL appear at locus DYS533 (dot line box)

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Figure 2: The electrophoregram of the father in the second pair using AGCU Y24 short tandem repeat kit. The results show that there are no allele peaks at locus DYS447, but duplicated allele peaks labeled 14 and OL are detected at locus DYS533 (dot line box)

Click here to view

There are two possibilities to explain these observations. The first is that the null type and the duplication are independent events. The null type is the result of mutations of primer binding sites, and duplication at the nearby Y-STR loci is generated by replication. The second explanation is that the “null allele” is a very long allele, and allele OL at the duplicated loci is from the null-type allele. The results of singleplex PCR and sequencing confirmed the second explanation. In the first father–son pair, an allele with a size of 358.08 bp was amplified with DYF387S1 primers and was located within the allelic size range of DYS533. In contrast, the locus DYS533 only showed one single allele (allele 12) in the marker size range of DYS533 [Figure 3]a. In addition, the sequencing results showed that at locus DYS387S1, allele 50 appeared with the repeat structure of [AAAG]3 GTAG[GAAG]4[AAAG]2 GAAG[AAAG]2[GAAG]11[AAAG]15[GAAG]11. These results prove that the OL peak represents a real DYF387S1 allele. A similar result was observed in the second father–son pair [Figure 3]a. In the allelic range of DYS19, only one allele sized 272.35 bp was amplified with singleplex DYS447 primers, and a single allele peak was shown in the size range of DYS19 [Figure 3]b. The genotype at DYS19 in the second pair was further confirmed by Yfiler ® Plus PCR Amplification Kit [Figure S1]. The sequencing result of the DYS447 locus confirmed allele 41 with the repeat structure of [TAATA]6[TAAAA]1[TAATA]12[TAAAA]1[TAATA]12[TAAAA]1[TAATA]8. The OL peak that appeared in the range of DYS19 represents a real DYS447 allele. Therefore, the long “null alleles” at DYF387S1 and DYS447 should be allele 50 and allele 41, respectively, designated by the number of repeats.[7] Both loci were located far outside the common allelic ladder ranges of 33–42 (DYF387S1) and 18–30 (DYS447) of the multiplex STR typing kits.
Figure 3: (a and b) The electrophoregram of singleplex polymerase chain reaction products. Plot A shows an allele with 358.08 bp at the locus DYF387S1 (bottom) and an allele 12 at locus DYS533 (top). Plot B shows an allele with 272.35 bp at the locus DYS447 (bottom) and an allele 14 at locus DYS19 (top)

Click here to view

Extremely long alleles have been observed at several other autosomal STR loci, such as SE33,[8] D3S1358,[5] D21S11,[3] D2S1338, and D19S433.[6] The long alleles can result from the variant allele consisting of a large number of repeat units.[3],[5],[6] A similar sequence pattern also occurred in the current two cases. Since the two long alleles went beyond the longest allele of the allelic ladder and very few intermediate alleles have been observed at DYF387S1 and DYS447 loci, the allele variants may arise from the Y-intrachromosomal nonallelic gene conversion.[8],[9],[10]

It is worth noting that DYF387S1 is located in the P1 palindrome of the Y-chromosome. In general, DYF387S1 is a duplicated marker and usually has two alleles (DYF387S1a/b) in males. However, only one allele was observed at the DYF387S1 locus in our first father–son pair. This phenomenon can be explained as a deletion of one allele or two alleles of the same size. Y-chromosome palindromes are known to consist of highly symmetrical inverted repeats.[10] The abundant gene conversion between palindromes can cause recombinant loss of heterozygosity (recLOH) (https://isogg.org/wiki/RecLOH). Therefore, it is more likely that DYF387S1 carries two alleles with the same number of repeats (55–55 alleles).

The migration of STR alleles to larger neighboring locus size ranges has previously been revealed with several commercial multiplex autosomal STR kits [3],[5],[6],[11],[12] but has been observed very rarely with Y-STR kits. In previously analyzed samples of 1160 father–son pairs, alleles outside the allelic ladder range were not found at any of the 42 Y-STR loci.[13] Therefore, the frequency of a long allele occurring at the Y-STR loci is <1/580.

In our two father–son pairs, both alleles at DYF387S1 and DYS447 shifted into the larger allelic size ranges of DYS533 and DYS19, respectively [Figure 1] and [Figure 2]. These ambiguous alleles can cause confusion when attempting to interpret data. Since mutations and deletions of primer binding sites or target regions have been documented, null alleles have been found at Y-STR loci.[14],[15] In this way, the absence of allele peaks at a locus allelic range may be mistyped as a null allele. In contrast, the overlapping mobilities can display double peaks at a locus size range that could potentially be misinterpreted as duplicated alleles due to the high frequency of gene conversion.[16],[17]

In our case, it is noteworthy that both sons inherited their abnormal Y-STR haplotypes from their fathers [Figure S2] and [Figure S3]. Therefore, the finding that the overlapping alleles migrated to the expected size ranges of the neighboring markers strengthened the evidence that the two Y-STR haplotypes had originated from the same paternal lineage.

  Conclusion Top

We have observed two extremely long alleles at DYF387S1 and DYS447. The ambiguous alleles migrated into the adjacent locus allelic ladder range when using multiplex Y-STR PCR kits. With an increasing number of loci being added to multiplex kits, similar phenomena should be observed more frequently, which can potentially impact on the interpretation of Y-STR profiles. Hence, re-amplification employing the same kit, additional amplification using alternative multiplex kits with different adjoining markers, or additional singleplex PCR amplification will be required to avoid incorrectly assigning the alleles falling outside the ladder range.


We would like to thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Kayser M. Forensic use of Y-chromosome DNA: A general overview. Hum Genet 2017;136:621-35.  Back to cited text no. 1
Butler JM, Butler JM. Fundamentals of Forensic DNA Typing. Amsterdam, Boston: Academic Press/Elsevier; 2010.  Back to cited text no. 2
Grubwieser P, Mühlmann R, Niederstätter H, Pavlic M, Parson W. Unusual variant alleles in commonly used short tandem repeat loci. Int J Legal Med 2005;119:164-6.  Back to cited text no. 3
Dauber EM, Dorner G, Wenda S, Schwartz-Jungl EM, Glock B, Bär W, et al. Unusual FGA and D19S433 off-ladder alleles and other allelic variants at the STR loci D8S1132, vWA, D18S51 and ACTBP2 (SE33). Forensic Sci Int Genet Suppl Series 2008;1:109-11.  Back to cited text no. 4
Raziel A, Oz C, Carmon AD, Ilsar R, Zamir A. Discordance at D3S1358 locus involving SGM plus™ and the European new generation multiplex kits. Forensic Sci Int Genet 2012;6:108-12.  Back to cited text no. 5
Dell'Ariccia-Carmon A, Raziel A, Oz C, Berdugo R, Zamir A. Long allele designations at D2S1338 and D19S433 loci as influenced by various multiplex STR kits. J Forensic Sci 2014;59:718-22.  Back to cited text no. 6
Gill P, Urquhart A, Millican E, Oldroyd N, Watson S, Sparkes R, et al. Anew method of STR interpretation using inferential logic – Development of a criminal intelligence database. Int J Legal Med 1996;109:14-22.  Back to cited text no. 7
Klein R, Braunschweiger G, Junge A, Wiegand P. A very long ACTBP2 (SE33) allele. Int J Legal Med 2003;117:235-6.  Back to cited text no. 8
Chen JM, Cooper DN, Chuzhanova N, Férec C, Patrinos GP. Gene conversion: Mechanisms, evolution and human disease. Nat Rev Genet 2007;8:762-75.  Back to cited text no. 9
Trombetta B, Cruciani F. Y chromosome palindromes and gene conversion. Hum Genet 2017;136:605-19.  Back to cited text no. 10
Jung JY, Kim EH, Oh YL, Park HC, Hwang JH, Lim SK, et al. Evaluation of forensic genetic parameters of 12 STR loci in the Korean population using the investigator ® HDplex kit. Int J Legal Med 2017;131:1247-9.  Back to cited text no. 11
Phillips C, Fernandez-Formoso L, Gelabert-Besada M, García-Magariños M, Amigo J, Carracedo A, et al. Global population variability in qiagen investigator HDplex STRs. Forensic Sci Int Genet 2014;8:36-43.  Back to cited text no. 12
Wu W, Ren W, Hao H, Nan H, He X, Liu Q, et al. Mutation rates at 42 Y chromosomal short tandem repeats in Chinese han population in Eastern China. Int J Legal Med 2018;132:1317-9.  Back to cited text no. 13
Budowle B, Aranda XG, Lagace RE, Hennessy LK, Planz JV, Rodriguez M, et al. Null allele sequence structure at the DYS448 locus and implications for profile interpretation. Int J Legal Med 2008;122:421-7.  Back to cited text no. 14
Turrina S, Caratti S, Ferrian M, De Leo D. Deletion and duplication at DYS448 and DYS626 loci: Unexpected patterns within the AZFc region of the Y-chromosome. Int J Legal Med 2015;129:449-55.  Back to cited text no. 15
Butler JM, Decker AE, Kline MC, Vallone PM. Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation. J Forensic Sci 2005;50:853-9.  Back to cited text no. 16
Butler JM. Y-Chromosome DNA testing. In: Butler JM, editor. Advanced Topics in Forensic DNA Typing: Methodology. Ch. 13. San Diego: Academic Press; 2012. p. 371-403.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3]


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