Paternally Inherited Trisomy at D21S11 and Mutation at DXS10135 Microsatellite Marker in a Case of Fetus Paternity Establishment

Egyptian Journal of Forensic Sciences (2015) xxx, xxx–xxx

HOSTED BY Contents lists available at ScienceDirect

Egyptian Journal of Forensic Sciences

journal homepage: http://www.journals.elsevier.com/egyptian-journal-of-forensic-sciences

CASE REPORT Paternally inherited trisomy at D21S11 and mutation at DXS10135 microsatellite marker in a case of fetus paternity establishment

Pankaj Shrivastava a,*, Toshi Jain a,b, Sonia Kakkar c, Veena Ben Trivedi a a DNA Fingerprinting Unit, State Forensic Science Laboratory, Department of Home (Police), Govt. of Madhya Pradesh, Sagar 470001, MP, India b School of Studies in Microbiology, Jiwaji University, Gwalior, MP, India c Deptt of Forensic Medicine, PGIMER, Chandigarh, India

Received 3 June 2015; revised 29 July 2015; accepted 6 September 2015

KEYWORDS Abstract The case of establishment of paternity of an aborted fetus was examined with 15 auto- DNA profiling; somal STR markers. The genotype of the fetus was X at amelogenin marker and showed inheritance D21S11; of both the alleles of father at D21S11 marker, thus displaying unusual tri-allelic pattern. The cases DXS10135; where mutation in any of biparental autosomal STR markers is observed, the use of additional STR X-STR; marker system is recommended. On testing all the three samples with 12 X-STR markers, all the Trisomy; maternal and paternal alleles were accounted in the female fetus except at DXS10135 marker. Mutation The genotypes of mother, fetus and father at DXS10135 were 20, 22; 20, 20 and 21, which confirmed mutation of the paternal allele in the female fetus. The paternal allele contracted from 21 to 20. The allele peak heights of D21S11 and DXS10135 markers were also examined to rule out the possibility of any false allele. The probability of paternity was 0.99999999, which confirmed the paternity of the fetus. This paper presents unusual occurrence of mutation observed with two multiplex STR systems, with AmpflSTR identifier plus Kit (Applied Biosystems, Foster City, CA) and Investigator Argus X-12 multiplex kit (Qiagen, Germany), thus suggesting forensic DNA experts on high alert while interpreting the DNA test results. Ó 2015 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences (IALFS).

1. Introduction are small sequences of DNA made up of repeating units of 2–6 nucleotides.1–3 Various STR based multiplexes have been The genetic analyses in paternity and in forensic casework are developed on autosomal and gonosomal markers which have based on the detection of short tandem repeats (STRs), which now become indispensable tools for determining individual identities and are being used widely in the forensic casework including paternity establishment, disasters and rarely in disor- * Corresponding author. Tel.: +91 9424371946, +91 7049154272. E-mail address: [email protected] (P. Shrivastava). der diagnostics. The analysis of STRs polymorphism located Peer review under responsibility of Forensic Medicine Authority. on the autosomal, X and Y chromosomes has been the focus http://dx.doi.org/10.1016/j.ejfs.2015.09.001 2090-536X Ó 2015 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences (IALFS).

Please cite this article in press as: Shrivastava P et al. Paternally inherited trisomy at D21S11 and mutation at DXS10135 microsatellite marker in a case of fetus paternity establishment, Egypt J Forensic Sci (2015), http://dx.doi.org/10.1016/j.ejfs.2015.09.001 2 P. Shrivastava et al. of much research in the field of population genetics, forensics 2. Material and methods and clinical genetics in recent years. STR markers located on autosomal chromosomes transmit to the next generation in Referral blood samples of mother and father along with an the Mendelian inheritance model where biological offspring aborted fetus were received as case exhibits for establishment shares one allele from father and the other from mother on of paternity of fetus to the DNA Fingerprinting Unit of State all the tested loci. While the X chromosome has a specific Forensic Science Laboratory, Sagar (MP) India, along with inheritance pattern of allele transfer where the women are identification and consent forms as per the guidelines of deciduous and follow mendelian inheritance while, men 4 DNA fingerprinting examination. DNA isolation was done are hemizygous and have the single X from the mother. The by Automated DNA extraction system 12 GC (PSS, Japan), Y-chromosome being inherited from the paternal side trans- using prefilled cartridges supplied by the manufacturer. Quan- ferred as such in all blood relative males of a family and so titative assessment was done by ABI 7000 Real Time PCR defines only patrilineages. (Applied Biosystems, Foster City, CA). Multiplexed PCR The chromosome number alterations are observed on the amplifications of the 15 autosomal STR Loci: D8S1179, autosomal chromosomes and are detected sometimes during D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, forensic DNA typing. Trisomy 21, commonly known as Down D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, syndrome (Fig. 1), is one of the best-recognized and most com- D5S818, FGA and Amelogenin were performed using the mon chromosome disorders, which is the cause of significant AmpflSTR identifiler plus Kit (Applied Biosystems, Foster mental retardation and physical anomalies. The incidence of City, CA) according to the manufacturer’s recommended pro- Down syndrome is approximately 1/800 newborns. An erred tocol. Multiplexed PCR amplifications of the 12 X STR Loci: combination of chromosomes at the time of oogenesis and DXS10103, DXS8378, DXS7132, DXS10134, DXS10074, spermatogenesis results in either one of them having an extra DXS10101, DXS10135, DXS7423, DXS10146, DXS10079, chromosome. So after the egg and sperm unite, the resulting HPRTB and DXS10148 and Amelogenin (sex determining cells will also have an altered number of chromosomes. In marker) were performed using the Investigator Argus X-12 about 95% of cases the extra chromosome 21 is of maternal PCR Amplification Kit (Qaigen) according to the manufac- origin, but, we present here a case with 21 trisomy from the turer’s recommended protocol. The PCR reagents have been father. We also report here mutation detected in two different standardized in the laboratory for consistency of results. STR multiplex systems, probably the first time, in which on PCR was performed using the half reaction volume of the genotyping of mother, father and female fetus showed trisomy manufacturer’s recommended protocol and using Gene Amp at D21S11 marker with the inheritance of both the paternal 9700 thermal cycler (Applied Biosystems, Foster City, CA). alleles with AmpflSTR identifier plus Kit (Applied Biosystems, The amplified PCR products were run on 3100 Genetic Foster City, CA) and mutation at DXS10135 marker on test- Analyzer (Applied Biosystems, Foster City, CA) following ing the same trio with Investigator Argus X-12 multiplex kit the recommended protocol. The samples were prepared using (Qiagen, Germany).

Figure 1 Results of Karyotyping showing 21 trisomy.

Table 1 Genotype of 15 autosomal STR markers in mother, child and father and paternity index.

*The paternal allele 30, 31 were inherited to the child.

Table 2 Genotype of 12 X-chromosomal STR m in mother, child and father with paternity index.

*The paternal allele 21 is mutated to 20 in the child.

10 lL of Hi-Di formamide (Applied Biosystems, Foster City, allele of father at D21S11 (Table 1 and Fig. 1) and paternal CA), with 0.125 lL size standard and 0.5 lL of the amplified allele mutation at DXS10135 STR markers (Table 2). The PCR product. The samples were denatured for 3 min at genotype at D21S11 marker in mother, fetus and father was 95 °C and snap cooled on ice before loading onto the instru- 28, 30; 28, 30, 31; 30, 31 respectively which confirmed trisomy ment. The analyzer was equipped with 36 cm arrays, with sep- 28, 30, 31 in child with the inheritance of both 30, 31 paternal aration of fragments using POP-4 polymer (Applied alleles (Fig. 2). The genotype at DXS10135 marker in mother, Biosystems, Foster City, CA) and the data were analyzed with fetus and father was 20, 22; 20, 20; 21 respectively confirmed Gene Mapper ID Analysis Software (Applied Biosystems, Fos- paternal allele mutation with contraction of allele from 21 to ter City, CA). A peak detection threshold of 50 RFUs was 20 (Fig. 3). The probabilities of paternity at autosomal and used for allele designation. X chromosomal STR markers were 0.99999999 and 0.99999 respectively establishing the reasonable degree of inclusion of 3. Results and discussion paternal alleles in the child. The 21 chromosome trisomy is a genetic disorder where the The DNA profiles of mother, female fetus and father were gen- child inherits two copies of chromosome from either parent. erated with 15 autosomal, 12 X chromosomal tetranucleotide The chance of inheritance is predominately from maternal side STR and gender specific amelogenin markers. The maternal as compared to paternal alleles. The chromosomal karyotyp- and paternal alleles at the examined STR markers were ing is the conventional and most widely used method of diag- unambiguously assigned to fetus except inheritance of both nosis of trisomy. The karyotyping detects the chromosomal

Figure 2 Paternity examination of child (fetus) with 15 autosomal STR markers.

aberration, but cannot reveal the inheritance pattern. The similar to each in heterozygous alleles (Table 3). The paternal DNA profiling method elaborates the genetic composition of inheritance is the rare event and this combination of genotype chromosomal trisomy with the inheritance pattern in a very is probably reported first time. There may be linkage of inher- short time. itance of autosomal and X chromosomal markers and this was The allele mutation is the other finding of this study. The observed in the present study. It is also important to notice additional STR markers were examined to support the pater- that marker DXS10135 in Investigator X 12 multiplex kit nal allele inclusion in the fetus on observation of trisomy at has been reported as most polymorphic marker in a recent D21S11 marker. The STR markers are prone to mutation study on Indian population.6 but it has been reported that longer alleles may undergo contraction and shorter alleles may undergo expansion due to 3.1. Quality control polymerase slippage with more prevalence from paternal side.5 The paternal allele at DXS10135 marker underwent one unit All the authors have passed proficiency testing and quality contraction from 21 to 20 tetra nucleotide repeats. The allele control exercise for DNA fingerprinting of GITAD, Spain peak heights of D21S11 and DXS10135 markers were also (http://gitad.ugr.es/ principal.htm). Also internal laboratory examined to rule out the possibility of any false allele and control standards were followed and controls provided with the peak height of relative fluorescence unit was also found multiplex kits used.

Figure 3 Paternity examination of child (fetus) with 12 X-chromosomal STR markers.

Table 3 The peak height in relative ﬂuorescence unit is similar Funding to each in heterozygous alleles. None declared.

Conﬂict of Interest

None declared.

*Showing inheritance of both the alleles from father on D21S11 and Acknowledgements mutation of paternal allele at DXS10135. The X STR kit used in the study was procured from the research project grant sanctioned from Madhya Pradesh Council of Science and Technology, Bhopal (MP) India. The authors are thankful to The Director, State Forensic Science 4. Conclusion Laboratory Sagar (MP) India for support.

This is a unique and rare report of inheritance of both References the alleles of father at D21S11 and simultaneous mutation of paternal allele at DXS10135 marker. The forensic scientists 1. Edwards A, Civitello A, Hammond HA, Caskey CT. DNA typing should be on very high alert while interpreting the genotyping and genetic mapping with trimeric and tetrameric tandem repeats. results. Am J Hum Genet 1991;49:746–56.

2. Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R. father and child due to maternal uniparental disomy 21 and a Genetic variation at ﬁve trimeric and tetrameric repeat loci in four mutation at the Y chromosome. Forensic Sci Int Genet 2009;3 human population groups. Genomics 1992;12(3):241–53. (2):141–3. 3. Ellegren H. Microsatellites: simple sequences with complex evolu- 6. Shrivastava P, Jain T, Gupta U, Trivedi VB. Genetic polymorphism tion. Nat Rev Genet 2004;5:435–45. study on 12 X STR loci of investigator Argus X STR kit in Bhil 4. Shrivastava P, Jain T, Trivedi VB. Usefulness of X STR Haplotype tribal population of Madhya Pradesh, India. Legal Med 2015;17 markers in Forensic DNA Proﬁling. Helix 2014;4:582–9. (3):214–7. 5. Lupo AM, Henke J, Henke L, Blank C, Ernsting A, Kozlowski P, et al. A paternity case with three genetic incompatibilities between