Non-invasive prenatal detection of trisomy 21 using tandem single nucleotide polymorphisms

Abstract

Background: Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA.

Methods and findings: Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects.

Conclusions: In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Conflict of interest statement

Competing Interests: Sujana Ghanta, Mary Ames, Mats Hidestrand, Mary Goetsch, and Craig Struble have been given stock options of Tandem Diagnostics, the company that is developing the technology the authors have reported in this study. Sujana Ghanta, Mary Ames, and Craig Struble currently serve as consultants to Tandem Diagnostics. Michael E. Mitchell is a board member, shareholder, and consultant of Tandem Diagnostics. Aoy Tomita-Mitchell is a shareholder of Tandem Diagnostics. Tandem Diagnostics also currently has a sponsored research with Dr. Aoy Tomita-Mitchell's and Michael E. Mitchell's labs at the Medical College of Wisconsin for research that is further development of this reported technology. Mats Hidestrand and Mary Goetsch work for Dr. Aoy Tomita-Mitchell's lab at the Medical College of Wisconsin and receive part of their salaries for this research. Michael E. Mitchell, William Thilly, and Aoy Tomita-Mitchell are also inventors on patent applications involving the technology reported herein. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Example of two tandem SNPs (SNP1, SNP2), which constitute 4 haplotypes (Haplotype1, Haplotype2, Haplotype3, Haplotype4).

Figure 2. Process overview.

DNA obtained from maternal buccal swab represent maternal germinal DNA. Tandem SNP sequences on chromosome 21 are amplified by MLA followed by High-Fidelity PCR (HiFi PCR) and CTCE analysis. DNA obtained from maternal plasma represents a mixture of fetal and maternal DNA. Tandem SNP sequences identified as heterozygous on maternal buccal swab are amplified on maternal plasma by MLA followed by High-Fidelity PCR (HiFi PCR) and CTCE analysis. CTCE analysis is followed by Tandem SNP evaluation to check for informativeness. Results with 3 peaks are subjected to HR analysis.

Figure 3. Theoretical CTCE electropherogram output from maternal buccal swab and plasma using tandem SNP analysis.

Sequences exhibiting three alleles are informative. White triangles represent DNA contributed by mother. Yellow triangles represent DNA contributed by the fetus. (A) and (B) Maternal plasma sample from a mother carrying a fetus with Disomy 21 (D21) (normal) is evident by three alleles (haplotypes) all having different areas where p3, the smaller peak, is equal to the difference between p1 and p2, resulting in an HR value = 1. (C) Maternal plasma sample from a mother carrying a fetus with Trisomy 21 is evident by two equal peaks with a third smaller peak suggesting non-disjunction occurred during meiosis I, resulting in an HR value = 0, or (D) three alleles with different areas if non-disjunction occurred during meiosis II where p1 or p2 is equal to twice the area of p3, resulting in HR = 2.

Figure 4. CTCE electropherogram and haplotype ratio calculations.

(A) and (B) each is a combination of two panels (top panel represents buccal swab and the bottom panel represents maternal plasma) and a table that shows the haplotype ratio calculation. Dotted line in the maternal plasma sample indicates the difference in peaks p1 and p2. (A) CTCE result of FDT0809 sample. Two peaks, p1 and p2 in the top panel (derived from a buccal swab), show that the subject/mother is germline heterozygous. Third peak (p3) seen between peaks p1 and p2 in the bottom panel (maternal plasma) is identified as the third haplotype, which is paternally inherited contribution to the fetus. As the experiment was performed in triplicate, HR values were reported as the mean +/− the standard deviation in the table below the panel. Peak areas (p1, p2, p3) are reported as mean value of the triplicates. The chromosome 21 call was D21 for this subject as the HR value is approximately 1. (B) CTCE result of FDT0805 sample. Two peaks, p1 and p2 in the top panel (buccal swab) show that the subject/mother is germline heterozygous. The chromosome 21 call is T21 for this subject as the HR value is approximately 2. Chromosome 21 calls by HR value were confirmed by fetal karyotype results.

Figure 5. Calculating yield for Multiplexed Linear Amplification (MLA).

A multiplexed linear PCR was setup with 12 primer sequences targeting 12 different targets including ten tandem SNP target sequences from chromosome 21, one target from chromosome 19, and one target on exon 1 of the REN gene. 2 ng genomic DNA from TK6 cells was used as template. (A) Mix 1a included template, buffer, and all primers but no polymerase was added and did not undergo linear amplification and denoted as a “before MLA” mix. (B) Mix 2a was identical to Mix 1a except for the addition of polymerase and 45 cycles of linear amplification and is denoted as an “after MLA” mix. One ul of each mix was then quantified for copy numbers of the REN gene target sequence by competitive PCR using an artificial mutant sequence spiked in at three different concentrations (101, 102, and 103 copies) followed by CTCE analysis. All “before MLA” and “after MLA” mixes were set up in triplicate (mix 1a, 1b, and 1c and did not include polymerase and did not undergo cycling, similarly, mix 2a, 2b, and 2c did include polymerase and did undergo cycling). All competitive PCR reactions were performed in triplicate for all six mixes. Comparison of REN gene target copy numbers before and after linear amplification divided by the number of cycles led to estimation of an average yield per cycle of linear amplification (Table S6 online).

Figure 6. Allelic bias is present following ligation-mediated PCR (LM-PCR) but not Multiplexed Linear Amplification (MLA).

(A) Starting from 6.25 ng genomic DNA, electropherogram of a heterozygous sequence at tandem SNP location rs11088086–rs2251447 following LM-PCR. Allele 2 was clearly preferentially amplified. (B) Starting from 6.25 ng genomic DNA, electropherogram of a heterozygous sequence at tandem SNP location rs11088086–rs2251447 following MLA. No allelic bias was observed for MLA with six assays.