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Trisomy 21 disclosure using STR and SNP markers typed by MiSeq FGx™ Forensic Genomics System

Published:September 30, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.09.023

      Abstract

      The presence of a tri-allelic pattern at a single locus in a multiplex short tandem repeat (STR) profile is a rarely observable event. Generally, based on peak height measured by the capillary electrophoresis (CE) method and combination of alleles, the tri-allelic pattern is distinguishable into two predominant types: type 1 and 2, which are caused, respectively, by somatic mutations and chromosomal rearrangements. When tri-allelic patterns at more than one STR located on the same chromosome are detected, there is a reasonable suspicion of a trisomy due to an extra copy of a chromosome. Therefore, information on the type of three-band pattern is usually limited to STRs localized on the same chromosome included in the forensic kit in use and sometimes in insufficient numbers to classify this event correctly. The opportunity to extend this evaluation to additional markers, such as SNPs detectable using NGS, has not yet been explored. In this study, using the ForenSeq™ DNA Signature Prep kit, two cases of autosomal aneuploidy were revealed on chromosome 21, relying not only on STRs assessment but also extending the analysis to the five identity-informative single nucleotide polymorphisms (iiSNPs) localized on chromosome 21.

      Keywords

      1. Introduction

      Autosomal short tandem repeats (STRs) are diploid loci that usually have no more than two identical (homozygous) or different (heterozygous) alleles per locus. Consequently, when autosomal STRs are genotyped by capillary electrophoresis (CE), one or at most two peaks per locus are expected in an electropherogram.
      The presence of a third peak is an unexpected event, only rarely detected, known as the tri-allelic pattern, which based on the peak height and the three alleles’ combination at the STR, has been classified into two predominant types: type 1, generally due to somatic mutations, and type 2, generated by chromosomal rearrangements (such as extensive localized chromosomal duplication or chromosomal aneuploidy) [
      • Clayton T.M.
      • Guest J.L.
      • Urquhart A.J.
      • et al.
      A genetic basis for anomalous band patterns encountered during DNA STR profiling.
      ].
      In the case of trisomy, in which an extra copy of a chromosome is present in the embryo due to the nondisjunction of the parents' chromosome, the type 2 pattern at the STR can manifest itself with height-balanced alleles but also with two alleles unbalanced in height or even just one allele that simulates false homozygosity [
      • Mansfield E.S.
      Diagnosis of down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms.
      ].
      In this instance, differentiating the type of tri-allelic pattern and the cause that generated it based exclusively on the assessment of the only one STR per chromosome included in the amplification kit used could be extremely difficult, making it necessary to resort to the typing of additional markers located on the chromosome affected by this event.
      This aim could be reached using more autosomal STRs amplification kits with several loci on the same chromosome or resorting to the massively parallel sequencing (MPS) technologies that enable typing more different loci simultaneously. The opportunity to extend this assessment to additional markers, such as SNPs detectable using MPS, has not yet been explored.
      Here are reported two cases of trisomy on chromosome 21 discovered through simultaneous typing by next-generation sequencing (NGS) technology of seven autosomal markers (two STRs and five identity-informative single nucleotide polymorphisms - iiSNPs) located on chromosome 21 and encompassed in the ForenSeq™ DNA Signature Prep kit.
      The examination led to the detection of type 2 tri-allelic pattern in correspondence with the two autosomal STRs and four of the five iiSNPs. Therefore, combining the information from STRs and iiSNPs, it was possible to recognize trisomy 21.

      2. Materials and methods

      The biological samples (amniotic fluid cells and chorionic villi) used in this study were collected after obtaining the entitled person's written informed consent under Italian law n.219/2017 and by approval from the University of Verona's research ethics committee review (CARU/CARP-12). The genomic DNA from the biological samples was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
      After DNA quantification with the Qubit dsDNA HS Assay Kit and the Qubit Fluorimeter (Qiagen), autosomal STRs amplification was performed with the GlobalFiler ™ IQC PCR Amplification Kit (Applied Biosystems, USA) and the PowerPlex® Fusion System (Promega Corp., Madison, WI). Amplicon typing was carried out by capillary electrophoresis technique using the SeqStudio Genetic Analyzer for HID and the ABI Prism 3130 Genetic Analyzer (Applied Biosystems), equipped with data analysis software GeneMapper® ID-X version 1.6 and 1.2 (Thermo Fisher, Waltham, MA), respectively.
      The same DNA samples were also analyzed by massively parallel sequencing using the Illumina MiSeq FGx Forensic Genomics system (San Diego, CA, USA). The DNA libraries were generated using the panel named DNA Primer Mix A - DPMA (59 STR + 94 iiSNP) included in the ForenSeq™ DNA Signature Prep kit (Verogen, San Diego, CA). The sequencing data was analyzed using ForenSeq Universal Analysis Software (UAS) v1.2 (Verogen), applying the default analytical threshold of 1.5% (minimum ≥10 reads) and interpretation threshold of 4.5% (minimum ≥30 reads).

      3. Results and discussion

      In a study aimed to assess the genotyping concordance between capillary electrophoresis (SeqStudio for HID and 3130 Genetic Analyzer – Applied Biosystems) and massively parallel sequencing (MiSeq FGx™ Forensic Genomics System - Illumina) techniques, DNA samples extracted from various biological matrices, including amniotic fluid cells and chorionic villus, were typed.
      Following DNA amplification with the GlobalFiler ™ IQC PCR Amplification kit and typing by capillary electrophoresis (SeqStudio Genetic Analyzer for HID), a type 2 tri-allelic pattern with balanced peak heights at the only one STR marker located on chromosome 21 (D21S11) included in the kit, was observed in the electropherogram of both samples, suggesting a located duplication event on the chromosome [Fig. 1A1 and A2].
      Fig. 1
      Fig. 1In two DNA samples, CE and MPS revealed type 2 tri-allelic patterns at the D21S11 and Penta D loci (A, B, and C), and only by MPS was that pattern evidenced at four of the five iiSNPs located on chromosome 21 (D).
      When DNA amplification was performed using the PowerPlex® Fusion System (Promega), which encompasses two STRs on chromosome 21 (D21S11 and Penta D), both samples exhibited a type 2 pattern at both loci. Based on the peak heights, at the D21S11 locus, was always distinguished a pattern of three peaks of equal height, while at the Penta D locus, in one sample, was observed a pattern with three even height alleles, and in the other sample, an unbalanced biallelic pattern in which one allele was twofold the height of the other (2:1) due to the overlap of two alleles of identical size, was revealed [Fig. 1B1 and B2].
      This event at the same two autosomal STRs was also confirmed by genotyping with the DNA Primer Mix A (DPMA) of the ForenSeq™ DNA Signature Prep kit using the MPS technique (Fig. 1C1 and C2).
      After the data analysis by ForenSeq AUS, in both samples, the loci D21S11 and Penta D were flagged with the icons of the quality control indicators referred to as "Allele Count" and "Imbalanced" that highlighted the presence of an extra allele or an imbalance of the read counts between two alleles, at a heterozygous locus.
      Since the ForenSeq™ DNA Signature Prep kit includes five identity-informative SNPs (rs722098, rs2830795, rs2831700, rs914165, and rs221956) located on chromosome 21, which could provide additional information on the pattern type, they have been considered in the evaluation. In one of the two samples, four of the five iiSNPs (rs722098, rs2830795, rs2831700, and rs914165) had the "imbalanced" icon assigned by ForenSeq AUS, showing biallelic patterns with unbalanced allelic read counts at each heterozygous locus, whereas one iiSNP (rs221956) was in homozygous. In the other sample, unbalanced allelic read counts to four heterozygous iiSNPs (rs2830795, rs2831700, rs914165, and rs221956) and one iiSNP (rs722098) in homozygous were revealed (Fig. 1D1 and D2).
      Based on the read counts, the iiSNP biallelic patterns exhibited an allelic intensity ratio of 2:1, probably due to the double presence of one of the two alleles in the heterozygous locus and therefore attributable to a type 2 tri-allelic pattern that includes two identical alleles.
      For the iiSNPs in homozygosity, since only one allelic variant was revealed, it was not possible to make a quantitative evaluation of the reads and was ruled uninformative, even though the presence of three identical allelic variants was theoretically conceivable.
      Therefore, it was reasonable upon these findings to infer that the type 2 tri-allelic patterns at STRs and iiSNPs observed in both samples were due to chromosomal aneuploidy.

      4. Conclusions

      The tri-allelic pattern occurrence at a marker is rare and, sometimes, could be tricky to distinguish which type it is when the evaluation is based only on the single locus per chromosome included in the multiplex STR system in use. The several markers analysis on the same chromosome could contribute to understanding if this event is of type 1 or 2.
      MPS enables simultaneous sequencing and genotyping of multiple STR and SNP loci in a single reaction. Here it is reported how using the ForenSeq™ DNA Signature Prep kit, which allows the parallel typing of two STRs and five iiSNPs on chromosome 21, has consented to classify as a type 2 tri-allelic pattern due to chromosomal rearrangements.
      What has been observed for trisomy 21 could also be applied to reveal other aneuploidies such as trisomies 13 and 18, where MPS analysis would allow analyzing, in addition to the single STR (D13S317 and D18S51) per chromosome present in the kit, further four iiSNPs located on chromosomes 13 and 18.

      Conflict of interest

      None.

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        A genetic basis for anomalous band patterns encountered during DNA STR profiling.
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