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 Table of Contents  
Year : 2016  |  Volume : 2  |  Issue : 2  |  Page : 115-118

A Case of Maternal Half-sisters Sharing Alleles at 18 X-chromosomal Short Tandem Repeat Loci

Department of Forensic Biology, Faculty of Forensic Medicine, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, P. R. China

Date of Web Publication16-Jun-2016

Correspondence Address:
De-Jian Lu
Faculty of Forensic Medicine, Zhongshan Medical School, Sun Yat.sen University, 74 Zhongshan 2nd. Road, Guangzhou 510080
P. R. China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2349-5014.184194

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Analysis of X-chromosome short tandem repeats (STRs) is very helpful in deficiency paternity testing. Here, we reported a case of kinship analysis that showed a potentially erroneous inclusion of paternal sisters between two women. The two women shared alleles at 18 X-chromosomal STR loci spanned from 14.76cM (DXS6807) to 184.19cM (DXS7423). When their relatives were not available for testing, biostatistical analysis for the 18 X-chromosomal STR loci and 24 autosomal STR loci revealed the most possible relationship between the two women was paternal sisters. However, when the father of one woman was available, the other father-daughter possibility was excluded. In the end, the likelihood ratio of STR marker and mitochondrial DNA (mtDNA) sequences confirmed the two women were maternal sisters. This case emphasizes a cautionary interpretation of X chromosomal marker in deficiency paternity cases with female offspring. Even though large parts of the X-chromosome haplotypes shared by two females, additional relatives and extended DNA typing (such as mtDNA) may be needed further to ascertain whether they are paternal or maternal sisters.

Keywords: Haplotype, kinship testing, recombination, X-chromosome short tandem repeat

How to cite this article:
Liu QL, Xue L, Zhao H, Lu DJ. A Case of Maternal Half-sisters Sharing Alleles at 18 X-chromosomal Short Tandem Repeat Loci. J Forensic Sci Med 2016;2:115-8

How to cite this URL:
Liu QL, Xue L, Zhao H, Lu DJ. A Case of Maternal Half-sisters Sharing Alleles at 18 X-chromosomal Short Tandem Repeat Loci. J Forensic Sci Med [serial online] 2016 [cited 2022 Dec 2];2:115-8. Available from: https://www.jfsmonline.com/text.asp?2016/2/2/115/184194

  Introduction Top

X-chromosomal short tandem repeat (X-STR) markers provide an extremely efficient tool in kinship testing, especially in deficiency paternity cases with female offspring or in maternity cases.[1],[2] As a consequence, a larger number of X-STRs have been developed for the forensic purpose,[3] and examples of deficiency cases solved by X-STRs had been illustrated in a few of papers.[2],[4],[5],[6],[7] However, it is noteworthy that recombination should be taken into account when genetic markers located on the same chromosome are analyzed simultaneously.[3],[8] For X-STR markers, recombination is limited to female meiosis. The coinheritance of two identical maternal X-chromosomes without recombination is very rare.[1] Sharing alleles of X-STR between two females usually indicates that they are paternal sisters. However, this report focused on a case in which the common alleles of 18 X-STR loci between the two sisters were inherited from the mother.

  Materials and Methods Top

Case introduction

Kinship testing was requested by two women: a 34-year-old putative sister (S1) and a 28-year-old sister (S2). S1 was adopted by a couple when she was a child; they wondered whether S1 was the elder sister of S2. In the first stage, only S1 and S2 were available for investigation. However, the puzzling disagreement between the pedigree likelihood of STR markers and mitochondrial DNA (mtDNA) sequencing lead to the father (F) of S2 taken part into the testing.

Sample and DNA extraction

After obtaining informed consent, oral swab samples were collected from the putative sister (S1), sister (S2), and father (F) of S2. Genomic DNA was extracted using the Chelex 100 method described by Walsh et al.[9]

X-short tandem repeat and autosomal short tandem repeat typing

The 18 X-STR loci were typed using two multiplex systems reported by us including MX 10-STR and MX 9-STR. MX 10-STR consisted of DXS7133, DXS6801, DXS981, DXS6809, DXS7424, DXS6789, DXS7132, GATA165B12, DXS101, and GATA31E08 in a single multiplex reaction, in which primer and polymerase chain reaction (PCR) conditions were as described.[10] MX 9-STR consisted of DXS6854, DXS9902, DXS6809, GATA172D05, HPRTB, DXS7423, DXS6807, DXS8378, and DXS8377 in a single multiplex reaction, in which primer and PCR conditions were as described.[11] The autosomal STR (AS-STR) loci included in PowerPlex ® 16 System Kit (Promega, Madison, WI, USA) and other non-CODIS loci were typed according to the manufacturer's instruction and our previous study.[12] The pedigree likelihood or likelihood ratio under different scenarios of disputed kinship for STR markers was calculated using the Mendel version 13.2 software [13] based on the allele frequencies described previously,[10],[11],[12] genetic distance from the website (http://www.chrx-str.org/) or recombination fractions as previous.[14]

Mitochondrial DNA typing

mtDNA for three hypervariable regions (HV-1, HV-2, and HV-3) was amplified as described by Parson et al.[15] and Paneto et al.,[16] respectively. PCR amplification was conducted on the GeneAmp PCR System 9700 (Applied Biosystem). Amplicon was purified using QIAquick PCR purification kit (Qiagen Inc.). Cycling sequencing of both strands was performed using BigDye ® Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems), using the same primers as in the PCR reaction. Reaction products were ethanol precipitated and electrophoresed in an ABI the 3130XL Genetic Analyzer. Sequences were aligned and compared with the revised Cambridge Reference Sequence [17],[18] using BioEdit version 7.2.5 software.[19]

  Results Top

The genotyping results of X-STR were showed in [Table 1]. The electrophoretogram of the MX 10-STR and MX 9-STR, genotypes of AS-STR, and sequencing results of mtDNA are provided as Supplementary file: Figures S1 and S2, Tables S1 and S2, respectively.
Table 1: The genetic localization and typing results of 18 X-chromosome short tandem repeat loci

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In the preliminary analysis, only the swab samples from S1 and S2 were submitted to laboratory. No parents of S1 or S2 were available for testing. Considering all possible relationships, the following mutually exclusive hypotheses [Figure 1] were subjected to biostatistical analysis:
Figure 1: Relationship hypotheses for S1 and S2

Click here to view

H1: S1 and S2 are full-sister.

H2: S1 and S2 are paternal half-sisters. They have the same father but a different mother.

H3: S1 and S2 are maternal half-sisters. They have the same mother but a different father.

H4: S1 and S2 are unrelated.

To select the most possible relationship, pedigree likelihoods were calculated for the above, different hypotheses [Table 2]. The results revealed that the most likely relationship between S1 and S2 was paternal half-sister because hypothesis H2 had the highest likelihood. It seemed that S1 and S2 shared the father. However, further investigations of mtDNA hypervariable regions could not exclude the possibility that S1 and S2 had the same mother because they had some identical sequences [Table S2]. Therefore, known close relative reference samples from S1 and S2 were requested to further ascertain the relationship. Finally, only the sample from the father (F) of S2 was available.
Table 2: Pedigree likelihood under different hypotheses (assuming linkage equilibrium) without genotype of father of S2

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When the typing results of STRs of the father (F) of S2 were taken into account, a father-daughter relationship between F and S1 was excluded for the condition in 13 X-STR loci [Table 1] and 10 AS-STRs [Table S1]. In this case, only maternal half-sister (H3) or unrelated (H4) relationship between S1 and S2 need to be considered. By incorporating the genotypes obtained from F with those of S1 and S2, a likelihood ratio of 1.7573 × 108 for ChrX-STR, a likelihood ratio of 3.5617 × 107 for AS-STR, and a combined likelihood ratio of 6.25876 × 1015 were obtained under the hypothesis that S1 and S2 have the same mother versus the hypothesis that S1 was unrelated to S2 (H3 vs. H4). Combined with the information of mtDNA sequences, it could be confirmed that S1 was the maternal half-sister of S2.

  Discussion Top

Analysis of X-chromosome STRs is very powerful in deficiency paternity testing. The whole X chromosome passes through the males to their daughters so the paternal X-chromosome haplotypes can be detected in their daughters.[2] If X-STR typing of two females yields identical X-chromosome haplotypes, it implies that they are very likely to be paternal sisters. In the present case, S1 and S2 share alleles at 18 X-STR loci which span 14.76–188.7 cM from the p-telomere to q-telomere.[8] Since the coinheritance of two identical maternal X chromosome without a recombination is rare,[1] S1 and S2 seemed to share the same paternal X chromosome when F was unavailable. The pedigree likelihood also demonstrated that the most possible relationship between them was paternal half-sisters. However, when the STR typing result was analyzed again with the genotypes of the father (F) of S2, the father-daughter relationship between F and S1 was excluded. It was therefore concluded that the matching haplotype between S1 and S2 only could be inherited from the same mother or unrelated females. Finally, considering the overall likelihood ratio of 6.25876 × 1015 for STR markers and identical information of mtDNA sequences between S1 and S2, a maternal half-sisterhood could be confirmed. In this case, analysis of the most likely haplotypes in the family indicated that a recombination had arisen between DXS9902 and DXS7132 with the probability of <0.135[1] [Figure 2].
Figure 2: The most likely haplotypes in the family. Recombination event in the mother was observed between DXS9902 and DXS7132. 0 indicates unknown phase

Click here to view

It was worth emphasizing that the mtDNA sequencing results lead to first consideration that S1 and S2 have the maternal half-sisterhood relationship, then additional relative reference sample was requested for testing. Finally, the paternal sisterhood between S1 and S2 was excluded by incorporating the typing results of the father of S2. Although analysis of X-chromosomal markers is very useful for solving paternity cases when the alleged father is unavailable, and only two sisters are under analysis,[1],[20] further investigations of other polymorphic DNA (such as mtDNA) seems important sometimes.

  Conclusion Top

The case reported here suggests that even though large parts of the X-chromosome haplotypes shared by two females, paternal sisterhood should be concluded cautiously if relatives are unavailable. Additional DNA typing from relatives of the putative females may play a vital role to ascertain whether they are paternal or maternal sisters. Despite the high likelihood of STR markers obtained by biostatistical analysis, the further investigation of mtDNA should not be ignored also.


This study was supported by the grant from the National Natural Science Foundation of China (Grant No. 81373245), and cultivation of Medical Young Teachers of College basic research of Sun Yat-sen University (14ykpy02). The authors are grateful to the voluntary

donors for their cases.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Szibor R. X-chromosomal markers: Past, present and future. Forensic Sci Int Genet 2007;1:93-9.  Back to cited text no. 2
Hering S, Edelmann J, Augustin C, Kuhlisch E, Szibor R. X chromosomal recombination – A family study analysing 39 STR markers in German three-generation pedigrees. Int J Legal Med 2010;124:483-91.  Back to cited text no. 3
Prieto-Fernández E, Baeta M, Núñez C, Zarrabeitia MT, Herrera RJ, Builes JJ, et al. Development of a new highly efficient 17 X-STR multiplex for forensic purposes. Electrophoresis 2016. Doi: 10.1002/elps.201500546. [Epub ahead of print].  Back to cited text no. 4
Hundertmark T, Hering S, Edelmann J, Augustin C, Plate I, Szibor R. The STR cluster DXS10148-DXS8378-DXS10135 provides a powerful tool for X-chromosomal haplotyping at Xp22. Int J Legal Med 2008;122:489-92.  Back to cited text no. 5
Li L, Lin Y, Liu Y, Zhu R, Zhao Z, Que T. A case of false mother included with 46 autosomal STR markers. Investig Genet 2015;6:9.  Back to cited text no. 6
Hering S, Augustin C, Edelmann J, Heidel M, Dressler J, Rodig H, et al. DXS10079, DXS10074 and DXS10075 are STRs located within a 280-kb region of Xq12 and provide stable haplotypes useful for complex kinship cases. Int J Legal Med 2006;120:337-45.  Back to cited text no. 7
Phillips C, Ballard D, Gill P, Court DS, Carracedo A, Lareu MV. The recombination landscape around forensic STRs: Accurate measurement of genetic distances between syntenic STR pairs using HapMap high density SNP data. Forensic Sci Int Genet 2012;6:354-65.  Back to cited text no. 8
Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 1991;10:506-13.  Back to cited text no. 9
Liu QL, Lu DJ, Li XG, Zhao H, Zhang JM, Lai YK, et al. Development of the nine X-STR loci typing system and genetic analysis in three nationality populations from China. Int J Legal Med 2011;125:51-8.  Back to cited text no. 10
Liu QL, Lu DJ, Wu WW, Hao HL, Chen YF, Zhao H. Genetic analysis of the 10 ChrX STRs loci in Chinese Han nationality from Guangdong province. Mol Biol Rep 2011;38:4879-83.  Back to cited text no. 11
Lu DJ, Liu QL, Zhao H. Genetic data of nine non-CODIS STRs in Chinese Han population from Guangdong Province, Southern China. Int J Legal Med 2011;125:133-7.  Back to cited text no. 12
Lange K, Papp JC, Sinsheimer JS, Sripracha R, Zhou H, Sobel EM. Mendel: The Swiss army knife of genetic analysis programs. Bioinformatics 2013;29:1568-70.  Back to cited text no. 13
Liu QL, Luo H, Zhao H, Huang XL, Cheng JD, Lu DJ. Recombination analysis of autosomal short tandem repeats in Chinese Han families. Electrophoresis 2014;35:883-7.  Back to cited text no. 14
Parson W, Parsons TJ, Scheithauer R, Holland MM. Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: Application of mtDNA sequence analysis to a forensic case. Int J Legal Med 1998;111:124-32.  Back to cited text no. 15
Paneto GG, Longo LV, Martins JA, de Camargo MA, Costa JC, de Mello AC, et al. Heteroplasmy in hair: Study of mitochondrial DNA third hypervariable region in hair and blood samples. J Forensic Sci 2010;55:715-8.  Back to cited text no. 16
Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Holland M, et al. DNA Commission of the International Society for Forensic Genetics: Guidelines for mitochondrial DNA typing. Int J Legal Med 2000;113:193-6.  Back to cited text no. 17
Parson W, Gusmão L, Hares DR, Irwin JA, Mayr WR, Morling N, et al. DNA Commission of the International Society for Forensic Genetics: Revised and extended guidelines for mitochondrial DNA typing. Forensic Sci Int Genet 2014;13:134-42.  Back to cited text no. 18
Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 1999;41:95-8.  Back to cited text no. 19
Castañeda M, Mijares V, Riancho JA, Zarrabeitia MT. Haplotypic blocks of X-linked STRs for forensic cases: Study of recombination and mutation rates. J Forensic Sci 2012;57:192-5.  Back to cited text no. 20


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]

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