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Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing
Meta Gene 24 (2020) 100658
Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing
T
Uthandaraman Mahalinga Rajaa, , Dhanapal Thilagaa, Nithyanandam Mahalakshmia, Teena Koshyb, Rajiv Rosec ⁎
a
DNA Division, Forensic Sciences Department, 30A, Kamarajar Salai, Mylapore, Chennai 600004, Tamil Nadu, India Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Porur, Chennai 600 116, Tamil Nadu, India c Molecular Diagnostics, PerkinElmer Health Sciences, Chennai 600 113, Tamil Nadu, India b
ARTICLE INFO
ABSTRACT
Keywords: D8S1179 Tri-allele Copy number variation Intrachromosomal duplication MLPA Prenatal BoBs
Short Tandem Repeat (STR) typing is routinely used in forensic casework, paternity disputes and in the detection of aneuploidies. Anomalous peak patterns are occasionally encountered in STR typing due to genetic variations, mutations and, other abnormalities. We report here a rare tri-allelic variant at the D8S1179 locus in a 6-monthold female child, observed during routine paternity testing in the Indian Tamil population. DNA eluted from the blood samples collected on FTA elute microcards were subjected to STR typing for autosomal and sex STR loci using the commercial AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Capillary electrophoresis was carried out in ABI 3130xl Genetic Analyzer and genotyped using GeneMapper ID-X Software v1.5. Copy number variations and aneuploidy status of chromosome 8 were analyzed using Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs assays. In this case of paternity exclusion, the child displayed a Type II tri-allelic pattern (10,13,14) at the D8S1179 locus. In addition, the mother displayed an abnormal 10,14 genotype (with heterozygous peak imbalance - 2:1) at the D8S1179 locus suggesting 10,10,14 pattern. MLPA and prenatal BoBs analyses did not detect any abnormalities on chromosome 8. These results suggest an intrachromosomal segmental duplication event at the D8S1179 locus (8q24.13) as the reason behind tri-allelic pattern and its inheritance. Understanding anomalous cases such as these can help in appropriate interpretation of the results in STR typing.
1. Introduction Polymorphic Short tandem Repeats (STR) analysis is widely used in forensic human identity testing (Butler, 2006) and in the diagnosis of aneuploidies (Schmidt et al., 2000). A battery of autosomal and sex chromosomal STR loci are used in forensic identity testing. These STRs follow general mendelian inheritance pattern in which the offspring inherits one allele from each parent. Thus, STR fragment analysis results display either a single peak denoting homozygosity or two peaks denoting heterozygosity at any particular locus. D8S1179 locus is a compound tetranucleotide repeat (repeat motif [TCTA][TCTG]), found at the chromosomal location 8q24.13, with reported alleles ranging from 7 to 19 repeats (Gettings et al., 2015). This region is also of medical importance as it influences the susceptibility to several solid tumors (Arakawa et al., 2017; Robbins et al., 2007)
Anomalous results such as off ladder peaks, tri-allelic variants and other abnormal peak patterns are occasionally observed during STR typing, which may be due to microvariants, somatic mutations, primer binding site mutations(Huel et al., 2007). Although rare, tri-allelic pattern can be detected at a single locus of a multiplex STR profile as an anomalous STR typing result. Data catalogued on the NIST STRBase (https://strbase.nist.gov/tri_tab.htm) shows 401 tri-allelic patterns encompassing all core STR loci, of which 22 tri-allelic patterns have been reported for D8S1179 locus. However, the data on tri-allelic inheritance of STR loci is sparse; A literature search revealed tri-allelic inheritance reports on D10S1248, D3S1358, D7S820 and TPOX loci alone (Jiang et al., 2016; Picanço et al., 2015; Vidal and Cassar, 2008; Xiao et al., 2019). Majority of the entries in STRbase are from the western and the Chinese population. This is the first report on the occurrence and inheritance of tri-allelic D8S1179 from the Indian Tamil population. Besides STR based fragment analysis, techniques such as Prenatal BACs on
Corresponding author. E-mail addresses: [email protected] (U.M. Raja), [email protected] (D. Thilaga), [email protected] (N. Mahalakshmi), [email protected] (T. Koshy), [email protected] (R. Rose). ⁎
https://doi.org/10.1016/j.mgene.2020.100658 Received 8 November 2019; Received in revised form 28 December 2019; Accepted 24 January 2020 Available online 24 January 2020 2214-5400/ © 2020 Elsevier B.V. All rights reserved.
Meta Gene 24 (2020) 100658
U.M. Raja, et al.
Beads (BOBs) and Multiplex ligation-dependent probe amplification (MLPA) were utilized in understanding the genetic basis of the triallelic pattern. Documentation of rare observations such as this, in paternity and forensic testing, is essential for understanding STR variants and their frequencies in different populations.
FBXO25 8p subtelomeric region MLPA probe
2. Materials and methods 2.1. DNA extraction and STR analysis Blood samples were collected from the subjects using FTA elute cards and DNA was extracted from the blood spots, according to the manufacturer's instructions (GE Healthcare Life Sciences, UK, Catalog no.: WB120410). The extracted DNA was then quantitated using qRTPCR and 1.0 ng of the extracted DNA was amplified using multiplexed PCR assay with commercial AmpFlSTR Identifiler Plus kit (Thermo Fisher Scientific, Waltham, MA, USA – Catalog no.: 4427368). The 15 autosomal STRs (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA) and the gender marker amelogenin were amplified and analyzed. The abnormal tri-allelic pattern at D8S1179 was reconfirmed using the Promega PowerPlex® Fusion kit (Promega, Madison, WI, USA – Catalog no: DC2402). Amplification reactions were carried out according to the manufacturer's instructions. The amplified products were then analyzed by capillary electrophoresis using Applied Biosystems ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and genotyped using GeneMapper ID-X Software v1.5. Appropriate controls were utilized during the amplification and the capillary electrophoresis steps to check for spurious/non specific amplifications/peaks.
8q23.3-2q24.11 region implicated in Langer gideon 8q24.13 location of D8S1179
ZC3H3 8q subtelomeric region MLPA probe Fig. 1. Schematic representation of Chromosome 8 and the locations of D8S1179 locus, Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs markers.
microdeletion regions (Rose et al., 2019). Prenatal BoBs procedure was carried out as per the manufacturer's instructions already described (Grati et al., 2015b). Briefly, samples were processed using respective kits, raw data were generated using Luminex200 (Luminex Inc.) and analysis was done with BoBSoftversion 2.0 (PerkinElmer).
2.2. Multiplex ligation-dependent probe amplification (MLPA) The subtelomeres were screened using the SALSA MLPA probemix P036 Subtelomeres Mix 1 containing 46 MLPA probes (2 probes for each chromosome). DNA specimens from 2 healthy individuals (male and female) were used as normal controls, and for all reactions, the manufacturer provided protocol was followed (“https://www.mlpa. com/WebForms/WebFormDBData.aspx?FileOID=_jWdp7NYd4Ag.,”). The fragments were analyzed on an Applied Biosystems 3730 capillary sequencer (Thermo Fisher Scientific Inc.) using Genescan 500 size standards (Thermo Fisher Scientific Inc.) and analysis was performed with Genescan software, v2.6.0. Individual peaks corresponding to each exon were identified based on the difference in migration relative to the size standards. The peak area of each fragment was compared with that of a control specimen. Using the GeneMarker software (SoftGenetics, LLC), 10 final probe ratios were determined by comparing the relative probe peak in the DNA specimen of interest to all reference specimens and generating a dosage quotient. A lower copy number of that exon will result in a decreased probe signal. A dosage quotient lower than 0.7 or higher than 1.3 indicate a heterozygous deletion or duplication, respectively (Bunyan et al., 2004).
3. Results The genetic markers used and their location on chromosome 8 is schematically represented in Fig. 1. STR typing results for D8S1179 locus in the biological mother and the child, using AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit, respectively, from the GeneMapper ID-X Software v1.5, are shown in Fig. 2A and B. Table 1 shows the genotype of the trio- Alleged father, child, and biological mother. 3.1. STR typing reveals a rare tri-allelic pattern at D8S1179 locus on chromosome 8 The alleged father was excluded from the paternity of the child based on the STR analysis of the blood samples using AmpFlSTR Identifiler Plus kit. The D8S1179 locus in the female child showed a triallelic genotype (10,13,14). In addition the biological mother displayed an abnormal biallelic genotype (2:1) at the D8S1179 locus. The results were reconfirmed using the Promega PowerPlex® Fusion kit.
2.3. Prenatal BoBs Prenatal BoBs is a ConformitéEuropéene (CE) marked in vitro diagnostic (IVD) bead based multiplex assay designed to detect aneuploidies of chromosomes, 13, 18, 21 and sex chromosomes, as well as gains and losses in 9 microdeletion syndrome regions (Cri Du Chat, DiGeorge, 10p14 deletion, Langer-Giedion, Miller–Dieker, Prader–Willi/ Angelman, Smith–Magenis, William–Beuren and Wolf Hirschhorn (Grati et al., 2015b, 2015a; Vialard et al., 2011). The analysis is based on BACs-on-Beads technology that utilizes the immobilization of probes from bacterial artificial chromosomes (BACs) on to fluorescently coded Beads (Luminex). This test is used as a rapid aneuploidy test in prenatal samples but with increased clinical sensitivity due to inclusion of the
3.2. MLPA analysis shows absence of chromosome 8 aneuploidy MLPA can be a reliable approach towards detection of aneuploidies. Abnormalities in the regions probed by MLPA will be reflected as variations in the dosage quotient compared to the reference samples. The obtained dosage quotient was within 0.7 and 1.3 (Bunyan et al., 2004) for all the chromosomes, indicating that the biological mother and the child do not harbor chromosome 8 aneuploidy (Fig. 3.). 2
Meta Gene 24 (2020) 100658
U.M. Raja, et al.
Fig. 2. Electropherogram of the tri-allelic pattern at the D8S1179 locus and the abnormal biallelic genotype (heterozygous peak imbalance) observed in the child and the mother, respectively, using AmpFlSTR Identifiler Plus kit (A) and Promega PowerPlex® Fusion kit (B).
3.3. Duplication of 8q24.13 is locus specific and doesn't extend into nearby 8q24.11
Table 1 STR profile of the alleged fatherbiological mother and the female child obtained with AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. STR Loci tested
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 D5S818 FGA D1S1656 D2S441 D10S1248 PENTA E PENTA D D12S391 D22S1045
Prenatal BoBs targets the 8q23.3-q24.11 as part of the Langer Giedion microdeletion region. For this sample, possible duplication was seen in the D8S1179 (8q24.13) locus. Due to the proximity of this locus to the Langer Giedion microdeletion region (8q23.3-q24.11), we processed this sample for Prenatal BoBs to understand if the duplication observed in the 8q24.13 extended into the 8q23.3-q24.11 region as well. The results indicated a normal diploid status for the Langer Giedion region in both the samples analyzed (Fig. 4.).
Genotype Alleged father
Female child
Biological mother
12,15 29,29 10,12 12,12 16,18 9,9.3 9,11 12,12 17,24 14,15 14,18 8,8 13,14 11,12 19,24 # # # # # # #
10,13,14 30,30.2 8,12 12,12 15,15 6,6 12,13 8,8 19,24 13,14 16,16 11,11 13,16 9,13 22,25 10,15 10,10 14,16 12,19 11,12 18,19 11,17
10,14 30,30 10,12 11,12 15,16 6,6 11,12 8,11 18,19 13,14 16,17 11,11 16,16 11,13 22,24 10,16 10,13 14,16 12,OL 12,12 19,23 11,11
4. Discussion In this article, we present a case of anomalous genotyping result, with a tri-allelic pattern observed at the D8S1179 locus. A case of paternity dispute was received at the Forensic Sciences Department, Chennai, Tamil Nadu, India. The results of the STR analysis showed a tri-allelic pattern at the D8S1179 locus (10,13,14 – three peaks with equal intensity) in the six-month-old female child and an abnormal biallelic genotype (10,14 –heterozygous peak imbalance with 2:1 ratio) in the mother. As this was a case of paternity exclusion, the data on the father's genotype was not available for comparison. The tri-allelic pattern was confirmed by using two commercially available STR kits (AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit), establishing that the observation was not a PCR artifact. In addition, the tri-allelic pattern was observed only in the D8S1179 locus and absent in the other loci tested, denoting that the profile is not a result of a mixture or contamination. Clayton et al., (Clayton et al., 2004) classified the different tri-allelic patterns in their seminal paper on tri-alleles. While Type I tri-allelic pattern is the commonly observed tri-allelic variant, Type II tri-allelic patterns are relatively rare. The three alleles of the D8S1179 locus in
STR markers in bold are common to both kits, STR markers in italics are present in Promega PowerPlex® Fusion kit only. Since this is a case of paternity exclusion, the alleged fathers sample was profiled using AmpFlSTR Identifiler Plus kit only.
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Meta Gene 24 (2020) 100658
U.M. Raja, et al.
Fig. 3. (A) MLPA results for the child's DNA as an electropherogram – a control sample A (blue peaks) is compared to those of the reference samples (red peak). The MLPA peak pattern of a DNA sample without genomic abnormalities will be identifcal to that of reference samples. (B) Analysis using GeneMarker – The dosage quotient (DQ) for each probe in each sample is calculated. A DQ below 0.7 or above 1.3 indicates a heterozygous deletion or duplication, respectively. Thus, no genetic abnormality is seen in the child sample.
Fig. 4. Prenatal BoBs assay results shows normal diploid complement for the Langer Giedion region (8q23.3-q24.11). All seven probes from the targeted region appears within the threshold for both the Male and Female references used.
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Meta Gene 24 (2020) 100658
U.M. Raja, et al.
Table 2 D8S1179 tri-allele Claytons's classification and frequency of observation from other populations reported in the literature. Population
No of tri-allelic patterns observed for D8S1179 locus
Sample size
Alleles
Clayton's classification (Clayton et al., 2004)
Reference
Bosnia and Serbia Chinese Belgian Indian Tamil
1 1 1 1
32800 NA 5964 9000
14/16/17 13/15/16 13/15/16 10/13/14
I II I II
(Huel et al., 2007) (Yang et al., 2015) (Mertens et al., 2009) Present study
Acknowledgements
this study were of equal intensity, fitting the Type II tri-allelic pattern described by Clayton et al. (Clayton et al., 2004). The frequency and Claytons' classification of D8S1179 tri-allele for various populations in the published literature are given in Table 2. The heterozygous peak imbalance (2:1) pattern at the D8S1179 locus in the mother was observed with both AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Heterozygous peak imbalance could arise due to the decrease in amplification in one of the alleles caused by mutations at primer binding site, trisomy or due to a tri-allele masked as a biallele genotype. The consistent observation of heterozygous peak imbalance with the Promega PowerPlex® Fusion System (which utilizes a different set of primers for the D8S1179 locus), suggests that the peak imbalance might not be due to primer binding site mutation but a double dose of one allele compared with the other. In addition, this 2:1 peak pattern conforms with Type II-B tri-allele subclassification as suggested by Picanco et al. (Picanço et al., 2015). The Claytons' Type II tri-allelic pattern can result from chromosomal aneuploidy or a localized intrachromosomal duplication (Mertens et al., 2009). To understand the ploidy status of chromosome 8 and to know if copy number variations (segmental duplications) are responsible for the observation of D8S1179 tri-allelic pattern, we used MLPA [8p subtelomeric region probe (FBXO25) and 8q subtelomeric region probe ZC3H3] and prenatal BoBs (probing the 8q23.3-2q24.11 region implicated in Langer Gideon syndrome). MLPA uses subtelomeric probes for p and q arms of all chromosomes (except the p arm of acrocentric chromosomes) and can detect chromosomal abnormalities including aneuploidies, while the prenatal bobs analysis included 7 probes for the 8q23.3-q24.11 region (Langer Gideon region). MLPA results showed absence of chromosome 8 aneuploidy, suggesting locus-specific intrachromosomal duplication event could be the reason for the tri-allelic pattern. Prenatal BoBs analysis revealed a normal diploid status for the Langer Giedion region (8q23.3-q24.11) showing that the duplication event doesn't extend into this region. On the basis of the results obtained we hypothesize that, in the biological mother, one of the two chromosome 8 could harbor two copies of D8S1179 (linked alleles 10 and 14). The child probably inherited the chromosome 8 with the two alleles for D8S1179 locus. Allele 13 (in the child) and allele 10 (in the biological mother) at the D8S1179 locus in the other chromosome 8 could explain the tri-allelic pattern (10,13,14 - seen in the child) and the abnormal heterozygous allelic imbalance (10,10,14 masked as 10,14 – seen in the biological mother). In conclusion, we report a rare type II tri-allelic inheritance observed in an Indian Tamil family. Anomalous results such as the triallelic pattern in this study can occur during routine STR typing. Although variants such as this do not affect the outcome of the analysis per se, they warrant a careful interpretation of the STR typing results. In addition, characterization of the such rare cases, using complementary molecular techniques, can shed light on possible origin of these abnormalities and can help in appropriate interpretations of the results obtained.
Routine work at the DNA Division, Forensic Sciences Department, Chennai, India, is supported by the Government of Tamil Nadu, India. We thank Dr. S. Karutha Pandian, Alagappa University, Tamil Nadu, India, for manuscript revision. References Arakawa, N., Sugai, T., Habano, W., Eizuka, M., Sugimoto, R., Akasaka, R., Toya, Y., Yamamoto, E., Koeda, K., Sasaki, A., Matsumoto, T., Suzuki, H., 2017. Genome-wide analysis of DNA copy number alterations in early and advanced gastric cancers. Mol. Carcinog. 56, 527–537. https://doi.org/10.1002/mc.22514. Bunyan, D.J., Eccles, D.M., Sillibourne, J., Wilkins, E., Thomas, N.S., Shea-Simonds, J., Duncan, P.J., Curtis, C.E., Robinson, D.O., Harvey, J.F., Cross, N.C.P., 2004. Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification. Br. J. Cancer 91, 1155–1159. https://doi.org/10.1038/sj.bjc.6602121. Butler, J.M., 2006. Genetics and genomics of core short tandem repeat loci used in human identity testing. J. Forensic Sci. 51, 253–265. https://doi.org/10.1111/j.1556-4029. 2006.00046.x. Clayton, T.M., Guest, J.L., Urquhart, A.J., Gill, P.D., 2004. A genetic basis for anomalous band patterns encountered during DNA STR profiling. J. Forensic Sci. 49, 1207–1214. Gettings, K.B., Aponte, R.A., Vallone, P.M., Butler, J.M., 2015. STR allele sequence variation: current knowledge and future issues. Forensic Sci. Int. Genet. 18, 118–130. https://doi.org/10.1016/j.fsigen.2015.06.005. Grati, F.R., Molina Gomes, D., Ferreira, J.C.P.B., Dupont, C., Alesi, V., Gouas, L., HorelliKuitunen, N., Choy, K.W., García-Herrero, S., de la Vega, A.G., Piotrowski, K., Genesio, R., Queipo, G., Malvestiti, B., Hervé, B., Benzacken, B., Novelli, A., Vago, P., Piippo, K., Leung, T.Y., Maggi, F., Quibel, T., Tabet, A.C., Simoni, G., Vialard, F., 2015a. Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies. Prenat. Diagn. 35, 801–809. https://doi.org/10.1002/pd. 4613. Grati, F.R., Vialard, F., Gross, S., 2015b. BACs-on-beadsTM (BoBsTM) assay for the genetic evaluation of prenatal samples and products of conception. Methods Mol. Biol. 1227, 259–278. https://doi.org/10.1007/978-1-4939-1652-8_13. https://www.mlpa.com/ WebForms/WebFormDBData.aspx?FileOID=_jWdp7NYd4Ag. Huel, R.L.M., Bašić, L., Madacki-Todorović, K., Smajlović, L., Eminović, I., Berbić, I., Miloš, A., Parsons, T.J., 2007. Variant alleles, Triallelic patterns, and point mutations observed in nuclear short tandem repeat typing of populations in Bosnia and Serbia. Croatian Med. J. 48, 494. Jiang, E., Pan, J., Han, M., Chen, L., Ma, Q., Wei, J., Huang, Y., Feng, S., Sun, Q., Xiao, P., Zheng, Z., 2016. Tri-allelic patterns at the D7S820 locus detected in two generations of a Chinese family. Int. J. Legal Med. 130, 23–26. https://doi.org/10.1007/s00414015-1166-6. Mertens, G., Rand, S., Jehaes, E., Mommers, N., Cardoen, E., De Bruyn, I., Leijnen, G., Van Brussel, K., Jacobs, W., 2009. Observation of tri-allelic patterns in autosomal STRs during routine casework. Forensic Sci. Int. 2, 38–40. https://doi.org/10.1016/j. fsigss.2009.07.005. Picanço, J.B., Raimann, P.E., da Motta, C.H.A.S., Rodenbusch, R., Gusmão, L., Alho, C.S., 2015. Identification of the third/extra allele for forensic application in cases with TPOX tri-allelic pattern. Forensic Sci. Int. Genet. 16, 88–93. https://doi.org/10.1016/ j.fsigen.2014.11.016. Robbins, C., Torres, J.B., Hooker, S., Bonilla, C., Hernandez, W., Candreva, A., Ahaghotu, C., Kittles, R., Carpten, J., 2007. Confirmation study of prostate cancer risk variants at 8q24 in African Americans identifies a novel risk locus. Genome Res. 17, 1717–1722. https://doi.org/10.1101/gr.6782707. Rose, R., Venkatesh, A., Pietilä, S., Jabeen, G., Jagadeesh, S.M., Seshadri, S., 2019. Utility and performance of bacterial artificial chromosomes-on-beads assays in chromosome analysis of clinical prenatal samples, products of conception and blood samples. J. Obstet. Gynaecol. Res. 45, 830–840. https://doi.org/10.1111/jog.13920. Schmidt, W., Jenderny, J., Hecher, K., Hackelöer, B.J., Kerber, S., Kochhan, L., Held, K.R., 2000. Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk. Mol. Hum. Reprod. 6, 855–860. https://doi.org/10. 1093/molehr/6.9.855. Vialard, F., Simoni, G., Aboura, A., De Toffol, S., Molina Gomes, D., Marcato, L., Serero, S., Clement, P., Bouhanna, P., Rouleau, E., Grimi, B., Selva, J., Gaetani, E., Maggi, F., Joseph, A., Benzacken, B., Grati, F.R., 2011. Prenatal BACs-on-beadsTM : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis. Prenat. Diagn. 31, 500–508. https://doi.org/10.1002/pd.2727. Vidal, C., Cassar, M., 2008. A case of tri-allelic pattern at locus D3S1358 on chromosome 3p21 inherited from paternal grandmother. Forensic Sci. Int. Genet. 2, 372–375.
Declaration of Competing Interest The authors declare that there is no conflict of interest. 5
Meta Gene 24 (2020) 100658
U.M. Raja, et al. https://doi.org/10.1016/j.fsigen.2008.06.002. Xiao, C., Wei, T., Huang, D., 2019. An unbalanced tri-allelic pattern at the D10S1248 locus: Characteristics, genetic basis and transmission. Legal Med. 41, 101627. https://doi.org/10.1016/j.legalmed.2019.101627.
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Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing
T
Uthandaraman Mahalinga Rajaa, , Dhanapal Thilagaa, Nithyanandam Mahalakshmia, Teena Koshyb, Rajiv Rosec ⁎
a
DNA Division, Forensic Sciences Department, 30A, Kamarajar Salai, Mylapore, Chennai 600004, Tamil Nadu, India Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Porur, Chennai 600 116, Tamil Nadu, India c Molecular Diagnostics, PerkinElmer Health Sciences, Chennai 600 113, Tamil Nadu, India b
ARTICLE INFO
ABSTRACT
Keywords: D8S1179 Tri-allele Copy number variation Intrachromosomal duplication MLPA Prenatal BoBs
Short Tandem Repeat (STR) typing is routinely used in forensic casework, paternity disputes and in the detection of aneuploidies. Anomalous peak patterns are occasionally encountered in STR typing due to genetic variations, mutations and, other abnormalities. We report here a rare tri-allelic variant at the D8S1179 locus in a 6-monthold female child, observed during routine paternity testing in the Indian Tamil population. DNA eluted from the blood samples collected on FTA elute microcards were subjected to STR typing for autosomal and sex STR loci using the commercial AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Capillary electrophoresis was carried out in ABI 3130xl Genetic Analyzer and genotyped using GeneMapper ID-X Software v1.5. Copy number variations and aneuploidy status of chromosome 8 were analyzed using Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs assays. In this case of paternity exclusion, the child displayed a Type II tri-allelic pattern (10,13,14) at the D8S1179 locus. In addition, the mother displayed an abnormal 10,14 genotype (with heterozygous peak imbalance - 2:1) at the D8S1179 locus suggesting 10,10,14 pattern. MLPA and prenatal BoBs analyses did not detect any abnormalities on chromosome 8. These results suggest an intrachromosomal segmental duplication event at the D8S1179 locus (8q24.13) as the reason behind tri-allelic pattern and its inheritance. Understanding anomalous cases such as these can help in appropriate interpretation of the results in STR typing.
1. Introduction Polymorphic Short tandem Repeats (STR) analysis is widely used in forensic human identity testing (Butler, 2006) and in the diagnosis of aneuploidies (Schmidt et al., 2000). A battery of autosomal and sex chromosomal STR loci are used in forensic identity testing. These STRs follow general mendelian inheritance pattern in which the offspring inherits one allele from each parent. Thus, STR fragment analysis results display either a single peak denoting homozygosity or two peaks denoting heterozygosity at any particular locus. D8S1179 locus is a compound tetranucleotide repeat (repeat motif [TCTA][TCTG]), found at the chromosomal location 8q24.13, with reported alleles ranging from 7 to 19 repeats (Gettings et al., 2015). This region is also of medical importance as it influences the susceptibility to several solid tumors (Arakawa et al., 2017; Robbins et al., 2007)
Anomalous results such as off ladder peaks, tri-allelic variants and other abnormal peak patterns are occasionally observed during STR typing, which may be due to microvariants, somatic mutations, primer binding site mutations(Huel et al., 2007). Although rare, tri-allelic pattern can be detected at a single locus of a multiplex STR profile as an anomalous STR typing result. Data catalogued on the NIST STRBase (https://strbase.nist.gov/tri_tab.htm) shows 401 tri-allelic patterns encompassing all core STR loci, of which 22 tri-allelic patterns have been reported for D8S1179 locus. However, the data on tri-allelic inheritance of STR loci is sparse; A literature search revealed tri-allelic inheritance reports on D10S1248, D3S1358, D7S820 and TPOX loci alone (Jiang et al., 2016; Picanço et al., 2015; Vidal and Cassar, 2008; Xiao et al., 2019). Majority of the entries in STRbase are from the western and the Chinese population. This is the first report on the occurrence and inheritance of tri-allelic D8S1179 from the Indian Tamil population. Besides STR based fragment analysis, techniques such as Prenatal BACs on
Corresponding author. E-mail addresses: [email protected] (U.M. Raja), [email protected] (D. Thilaga), [email protected] (N. Mahalakshmi), [email protected] (T. Koshy), [email protected] (R. Rose). ⁎
https://doi.org/10.1016/j.mgene.2020.100658 Received 8 November 2019; Received in revised form 28 December 2019; Accepted 24 January 2020 Available online 24 January 2020 2214-5400/ © 2020 Elsevier B.V. All rights reserved.
Meta Gene 24 (2020) 100658
U.M. Raja, et al.
Beads (BOBs) and Multiplex ligation-dependent probe amplification (MLPA) were utilized in understanding the genetic basis of the triallelic pattern. Documentation of rare observations such as this, in paternity and forensic testing, is essential for understanding STR variants and their frequencies in different populations.
FBXO25 8p subtelomeric region MLPA probe
2. Materials and methods 2.1. DNA extraction and STR analysis Blood samples were collected from the subjects using FTA elute cards and DNA was extracted from the blood spots, according to the manufacturer's instructions (GE Healthcare Life Sciences, UK, Catalog no.: WB120410). The extracted DNA was then quantitated using qRTPCR and 1.0 ng of the extracted DNA was amplified using multiplexed PCR assay with commercial AmpFlSTR Identifiler Plus kit (Thermo Fisher Scientific, Waltham, MA, USA – Catalog no.: 4427368). The 15 autosomal STRs (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA) and the gender marker amelogenin were amplified and analyzed. The abnormal tri-allelic pattern at D8S1179 was reconfirmed using the Promega PowerPlex® Fusion kit (Promega, Madison, WI, USA – Catalog no: DC2402). Amplification reactions were carried out according to the manufacturer's instructions. The amplified products were then analyzed by capillary electrophoresis using Applied Biosystems ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and genotyped using GeneMapper ID-X Software v1.5. Appropriate controls were utilized during the amplification and the capillary electrophoresis steps to check for spurious/non specific amplifications/peaks.
8q23.3-2q24.11 region implicated in Langer gideon 8q24.13 location of D8S1179
ZC3H3 8q subtelomeric region MLPA probe Fig. 1. Schematic representation of Chromosome 8 and the locations of D8S1179 locus, Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs markers.
microdeletion regions (Rose et al., 2019). Prenatal BoBs procedure was carried out as per the manufacturer's instructions already described (Grati et al., 2015b). Briefly, samples were processed using respective kits, raw data were generated using Luminex200 (Luminex Inc.) and analysis was done with BoBSoftversion 2.0 (PerkinElmer).
2.2. Multiplex ligation-dependent probe amplification (MLPA) The subtelomeres were screened using the SALSA MLPA probemix P036 Subtelomeres Mix 1 containing 46 MLPA probes (2 probes for each chromosome). DNA specimens from 2 healthy individuals (male and female) were used as normal controls, and for all reactions, the manufacturer provided protocol was followed (“https://www.mlpa. com/WebForms/WebFormDBData.aspx?FileOID=_jWdp7NYd4Ag.,”). The fragments were analyzed on an Applied Biosystems 3730 capillary sequencer (Thermo Fisher Scientific Inc.) using Genescan 500 size standards (Thermo Fisher Scientific Inc.) and analysis was performed with Genescan software, v2.6.0. Individual peaks corresponding to each exon were identified based on the difference in migration relative to the size standards. The peak area of each fragment was compared with that of a control specimen. Using the GeneMarker software (SoftGenetics, LLC), 10 final probe ratios were determined by comparing the relative probe peak in the DNA specimen of interest to all reference specimens and generating a dosage quotient. A lower copy number of that exon will result in a decreased probe signal. A dosage quotient lower than 0.7 or higher than 1.3 indicate a heterozygous deletion or duplication, respectively (Bunyan et al., 2004).
3. Results The genetic markers used and their location on chromosome 8 is schematically represented in Fig. 1. STR typing results for D8S1179 locus in the biological mother and the child, using AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit, respectively, from the GeneMapper ID-X Software v1.5, are shown in Fig. 2A and B. Table 1 shows the genotype of the trio- Alleged father, child, and biological mother. 3.1. STR typing reveals a rare tri-allelic pattern at D8S1179 locus on chromosome 8 The alleged father was excluded from the paternity of the child based on the STR analysis of the blood samples using AmpFlSTR Identifiler Plus kit. The D8S1179 locus in the female child showed a triallelic genotype (10,13,14). In addition the biological mother displayed an abnormal biallelic genotype (2:1) at the D8S1179 locus. The results were reconfirmed using the Promega PowerPlex® Fusion kit.
2.3. Prenatal BoBs Prenatal BoBs is a ConformitéEuropéene (CE) marked in vitro diagnostic (IVD) bead based multiplex assay designed to detect aneuploidies of chromosomes, 13, 18, 21 and sex chromosomes, as well as gains and losses in 9 microdeletion syndrome regions (Cri Du Chat, DiGeorge, 10p14 deletion, Langer-Giedion, Miller–Dieker, Prader–Willi/ Angelman, Smith–Magenis, William–Beuren and Wolf Hirschhorn (Grati et al., 2015b, 2015a; Vialard et al., 2011). The analysis is based on BACs-on-Beads technology that utilizes the immobilization of probes from bacterial artificial chromosomes (BACs) on to fluorescently coded Beads (Luminex). This test is used as a rapid aneuploidy test in prenatal samples but with increased clinical sensitivity due to inclusion of the
3.2. MLPA analysis shows absence of chromosome 8 aneuploidy MLPA can be a reliable approach towards detection of aneuploidies. Abnormalities in the regions probed by MLPA will be reflected as variations in the dosage quotient compared to the reference samples. The obtained dosage quotient was within 0.7 and 1.3 (Bunyan et al., 2004) for all the chromosomes, indicating that the biological mother and the child do not harbor chromosome 8 aneuploidy (Fig. 3.). 2
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Fig. 2. Electropherogram of the tri-allelic pattern at the D8S1179 locus and the abnormal biallelic genotype (heterozygous peak imbalance) observed in the child and the mother, respectively, using AmpFlSTR Identifiler Plus kit (A) and Promega PowerPlex® Fusion kit (B).
3.3. Duplication of 8q24.13 is locus specific and doesn't extend into nearby 8q24.11
Table 1 STR profile of the alleged fatherbiological mother and the female child obtained with AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. STR Loci tested
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 D5S818 FGA D1S1656 D2S441 D10S1248 PENTA E PENTA D D12S391 D22S1045
Prenatal BoBs targets the 8q23.3-q24.11 as part of the Langer Giedion microdeletion region. For this sample, possible duplication was seen in the D8S1179 (8q24.13) locus. Due to the proximity of this locus to the Langer Giedion microdeletion region (8q23.3-q24.11), we processed this sample for Prenatal BoBs to understand if the duplication observed in the 8q24.13 extended into the 8q23.3-q24.11 region as well. The results indicated a normal diploid status for the Langer Giedion region in both the samples analyzed (Fig. 4.).
Genotype Alleged father
Female child
Biological mother
12,15 29,29 10,12 12,12 16,18 9,9.3 9,11 12,12 17,24 14,15 14,18 8,8 13,14 11,12 19,24 # # # # # # #
10,13,14 30,30.2 8,12 12,12 15,15 6,6 12,13 8,8 19,24 13,14 16,16 11,11 13,16 9,13 22,25 10,15 10,10 14,16 12,19 11,12 18,19 11,17
10,14 30,30 10,12 11,12 15,16 6,6 11,12 8,11 18,19 13,14 16,17 11,11 16,16 11,13 22,24 10,16 10,13 14,16 12,OL 12,12 19,23 11,11
4. Discussion In this article, we present a case of anomalous genotyping result, with a tri-allelic pattern observed at the D8S1179 locus. A case of paternity dispute was received at the Forensic Sciences Department, Chennai, Tamil Nadu, India. The results of the STR analysis showed a tri-allelic pattern at the D8S1179 locus (10,13,14 – three peaks with equal intensity) in the six-month-old female child and an abnormal biallelic genotype (10,14 –heterozygous peak imbalance with 2:1 ratio) in the mother. As this was a case of paternity exclusion, the data on the father's genotype was not available for comparison. The tri-allelic pattern was confirmed by using two commercially available STR kits (AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit), establishing that the observation was not a PCR artifact. In addition, the tri-allelic pattern was observed only in the D8S1179 locus and absent in the other loci tested, denoting that the profile is not a result of a mixture or contamination. Clayton et al., (Clayton et al., 2004) classified the different tri-allelic patterns in their seminal paper on tri-alleles. While Type I tri-allelic pattern is the commonly observed tri-allelic variant, Type II tri-allelic patterns are relatively rare. The three alleles of the D8S1179 locus in
STR markers in bold are common to both kits, STR markers in italics are present in Promega PowerPlex® Fusion kit only. Since this is a case of paternity exclusion, the alleged fathers sample was profiled using AmpFlSTR Identifiler Plus kit only.
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Fig. 3. (A) MLPA results for the child's DNA as an electropherogram – a control sample A (blue peaks) is compared to those of the reference samples (red peak). The MLPA peak pattern of a DNA sample without genomic abnormalities will be identifcal to that of reference samples. (B) Analysis using GeneMarker – The dosage quotient (DQ) for each probe in each sample is calculated. A DQ below 0.7 or above 1.3 indicates a heterozygous deletion or duplication, respectively. Thus, no genetic abnormality is seen in the child sample.
Fig. 4. Prenatal BoBs assay results shows normal diploid complement for the Langer Giedion region (8q23.3-q24.11). All seven probes from the targeted region appears within the threshold for both the Male and Female references used.
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Table 2 D8S1179 tri-allele Claytons's classification and frequency of observation from other populations reported in the literature. Population
No of tri-allelic patterns observed for D8S1179 locus
Sample size
Alleles
Clayton's classification (Clayton et al., 2004)
Reference
Bosnia and Serbia Chinese Belgian Indian Tamil
1 1 1 1
32800 NA 5964 9000
14/16/17 13/15/16 13/15/16 10/13/14
I II I II
(Huel et al., 2007) (Yang et al., 2015) (Mertens et al., 2009) Present study
Acknowledgements
this study were of equal intensity, fitting the Type II tri-allelic pattern described by Clayton et al. (Clayton et al., 2004). The frequency and Claytons' classification of D8S1179 tri-allele for various populations in the published literature are given in Table 2. The heterozygous peak imbalance (2:1) pattern at the D8S1179 locus in the mother was observed with both AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Heterozygous peak imbalance could arise due to the decrease in amplification in one of the alleles caused by mutations at primer binding site, trisomy or due to a tri-allele masked as a biallele genotype. The consistent observation of heterozygous peak imbalance with the Promega PowerPlex® Fusion System (which utilizes a different set of primers for the D8S1179 locus), suggests that the peak imbalance might not be due to primer binding site mutation but a double dose of one allele compared with the other. In addition, this 2:1 peak pattern conforms with Type II-B tri-allele subclassification as suggested by Picanco et al. (Picanço et al., 2015). The Claytons' Type II tri-allelic pattern can result from chromosomal aneuploidy or a localized intrachromosomal duplication (Mertens et al., 2009). To understand the ploidy status of chromosome 8 and to know if copy number variations (segmental duplications) are responsible for the observation of D8S1179 tri-allelic pattern, we used MLPA [8p subtelomeric region probe (FBXO25) and 8q subtelomeric region probe ZC3H3] and prenatal BoBs (probing the 8q23.3-2q24.11 region implicated in Langer Gideon syndrome). MLPA uses subtelomeric probes for p and q arms of all chromosomes (except the p arm of acrocentric chromosomes) and can detect chromosomal abnormalities including aneuploidies, while the prenatal bobs analysis included 7 probes for the 8q23.3-q24.11 region (Langer Gideon region). MLPA results showed absence of chromosome 8 aneuploidy, suggesting locus-specific intrachromosomal duplication event could be the reason for the tri-allelic pattern. Prenatal BoBs analysis revealed a normal diploid status for the Langer Giedion region (8q23.3-q24.11) showing that the duplication event doesn't extend into this region. On the basis of the results obtained we hypothesize that, in the biological mother, one of the two chromosome 8 could harbor two copies of D8S1179 (linked alleles 10 and 14). The child probably inherited the chromosome 8 with the two alleles for D8S1179 locus. Allele 13 (in the child) and allele 10 (in the biological mother) at the D8S1179 locus in the other chromosome 8 could explain the tri-allelic pattern (10,13,14 - seen in the child) and the abnormal heterozygous allelic imbalance (10,10,14 masked as 10,14 – seen in the biological mother). In conclusion, we report a rare type II tri-allelic inheritance observed in an Indian Tamil family. Anomalous results such as the triallelic pattern in this study can occur during routine STR typing. Although variants such as this do not affect the outcome of the analysis per se, they warrant a careful interpretation of the STR typing results. In addition, characterization of the such rare cases, using complementary molecular techniques, can shed light on possible origin of these abnormalities and can help in appropriate interpretations of the results obtained.
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Declaration of Competing Interest The authors declare that there is no conflict of interest. 5
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