Base compositions of 12 amplicons in HV1 derived from primers covering coordinates15924-16428 and base compositions of 12 amplicons in HV2 derived from primers covering coordinates 31-576 were determined using ESI-TOF-MS based PLEX-ID system. Eight segments in HV1 and ten segments in HV2 were found to be polymorphic. Highly polymorphic information was available in the regions of 16124- 16250 and 138-340. After the assay was applied to a special maternity duo case, the false mother was excluded as the biological mother despite the fact that they shared at least an allele at 46 autosomal STR loci.

•    mtDNA
•    ESI-TOF-MS
•    PLEX-ID
•    Forensic genetics

Li L, Liu Y, Lin Y, Zhao Z (2015) PCR/ESI-TOF-MS Based Method for Detecting Polymorphisms of mtDNA in Chinese Han Population and Testing of a Special Maternity Case. Ann Forensic Res Anal 2(2): 1018.

Due to maternal inheritance, mitochondrial DNA (mtDNA) is valuable for testing of special kinship, e.g. relationships between maternal individuals, half-sibling sisters, aunt-nephew, maternal uncle-nephew and grandmother-granddaughter. In addition, mtDNA can be used for detection of problematic samples such as old and degraded bones, teeth and hair shafts. Identification of the remains of Russia’s last Tsar Nicholas II is a typical case about mtDNA application [1].

In forensic laboratories, mtDNA detection was routinely performed by Sanger sequencing, but the technique was only for analysis of part of mtDNA hypervariable regions (16024-16365 in HV1 and 73-340 in HV2). If the technology of higher resolving power can be used, more information about mtDNA in wider range will be gathered. In this study, multiplex PCR amplification followed by electrospray Ionisation time-of-flight mass spectrometry (ESITOF MS) was used to detect the polymorphisms of mtDNA by characterizing the base compositions of HV1 and HV2, nucleotide positions 15924-16428 [Figure 1] and 31-576 [Figure 2] [2], respectively. This assay allows for maximum discrimination of the hypervariable segments of mtDNA without targeting specific nucleotide positions.

Sample collection

Blood samples were collected from twelve unrelated Chinese Han individuals with informed consent.

DNA extraction and quantification

Genomic DNA was extracted from blood samples using QIAamp Mini Blood kit (Qiagen, German) and quantified using the Quantifiler Human DNA Quantification kit (Life Technologies, USA) and then the DNA concentration was adjusted to 10ng/µL. mtDNA amplification and detection American Abbott company provided technical service for the mtDNA analysis [3,4]. Primer sequences were reported by Hall TA et al in 2009 [5]. Firstly, 8 triplex PCR systems were used to amplify 12 segments of mtDNA HV1 coordinates 15924-16428 and 12 segments of HV2 coordinates 31-576 (the adjacent segments overlap 2-22 bp, shown in [Figure 3]. Then the PCR products were separated by ESI-TOF MS and the base compositions of HV1 and HV2 regions were determined by IBIS Track software (Abbott, USA) on the PLEX-ID platform.

During the test, known sample which had been sequenced was used as positive control. PCR amplifications and clean-up were conducted in Mastercycler (Eppendorf, German) on 96-well autosomal STRs detected, but she was adamant that she had not given birth to the child. In order to define the facts, mtDNA and SNP detection was followed.

Techniques for mtDNA detection

Various technologies for mtDNA detection have been reported, such as Sanger sequencing, high-resolution melt (HRM) profiling, denaturing high-performance liquid chromatography (DHPLC), exo-DNA polymerase and RFLP analysis, single strand conformation polymorphism (SSCP) analysis, denaturing gradient Gel electrophoresis assay, pyrosequencing, SNapShot and nextgeneration sequencing. For example, detection of mitochondrial heteroplasmy in cerebrospinal fluid using DHPLC was reported by Conley in 2003 [6]; Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R) (exo-) DNA polymerase and RFLP analysis was reported by Jacobi in 2001 [7]. In 1997 Nollau found that SSCP analysis could be used for detecting heteroplasmic mtDNA if the level of heteroplasmy was above 10% [8]; In 2000 Tully confirmed that the sensitivity of denaturing gradient Gel electrophoresis assay was about 1% [9]. It was worth mentioning that above techniques were only for detection of part of mtDNA hypervariable regions (16024- 16365 in HV1 and 73-340 in HV2) because of the limitation of discrimination power.

In this work we applied ESI-TOF-MS assay on the PLEX-ID platform and more discriminating information than that from Sanger sequencing was obtained. ESI-TOF-MS assay for HV1 and HV2 were designed to read bases 15924-16428 and 31- 576 respectively, while the Sanger sequencing for HV1 and HV2 were designed to read bases 16024-16365 and 73-340, which means that ESI-TOF-MS assay spans regions larger than Sanger sequencing. Although the PLEX-ID platform for analyzing of mtDNA is now no longer available, the comparative analysis of the ESI-TOF-MS technology and the traditional fluorescencebased capillary electrophoresis for typing of amplicons has demonstrated the potential advantages of the mass-spectrometric technique [10].

Polymorphism of mtDNA HV1

8 out of 12 segments of HV1 (15924-16428) showed SNP and/ or poly-C length polymorphisms [Table 1]. It could be seen that 16124-16250 in HV1 was high informative for forensic purpose.

Polymorphism of mtDNA HV2

10 out of 12 segments of HV2 (31-576) showed SNP and/or poly-C length polymorphisms [Table 2]. It was found that 138- 340 in HV2 was high polymorphic

Case application

The assay was applied to the special duo case in which the false mother could not be excluded as the biological mother because they shared at least an allele at 46 STR loci. To reach accurate conclusion, mtDNA detection was carried out as supplement to autosomal STR loci. mtDNA profiles clearly excluded the mother as the biological parent because the two persons had clear differences at 9 of the 24 fragments [Table 3]. The conclusion was confirmed by additional typing of SNP [Table 4]. Later on, investigation confirmed that the putative mother was in fact the boy’s paternal aunt. The mtDNA detection results meant that the mass spectrometry-based method for mitochondrial profiling could have promising prospects.

We would like to acknowledge Abbott company for providing the PLEX-ID platform and supplying the Forensic Mitochondria kits. We would also like to thank Dr. Steve Hofstadler and Dr. Tom Hall for their assistance with identifying the DNA samples on PLEX-ID.

1. Gill P, Ivanov PL, Kimpton C, Piercy R, Benson N, Tully G, et al. Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 1994; 6: 130-135.

2. Homosapiens mitochondrion, complete genome. Table 4: SNP differences between the detected mother and boy. SNP locus Mother(M) Boy(B) rs 1109037 A G rs 214955 G A rs 7229946 G A rs 985492 C T rs 9951171 G A

3. Howard R, Encheva V, Thomson J, Bache K, Chan YT, Cowen S, et al. Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry. Forensic Sci Int Genet. 2013; 7: 1-9

4. Ivanov PL. A new approach to forensic medical typing of human mitochondrial DNA with the use of mass-spectrometric analysis of amplified fragments: PLEX-ID automated genetic analysis system. Sud Med Ekspert. 2010; 53: 46-51.

5. Hall TA, Sannes-Lowery KA, McCurdy LD, Fisher C, Anderson T, Henthorne A, et al. Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automatedelectrospray ionization mass spectrometry. Anal Chem. 2009; 81: 7515-7526.

6. Conley YP, Brockway H, BeattyM, Kerr ME. Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography. Brain Res Proto. 2003; 12: 99-103.

7. Jacobi F K, Meyer J, Pusch C M, Wissinger B. Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis. Mutat Res. 2001; 478: 141-151.

8. Nollau P, Wagener C. Methods for detection of point mutations: performance and quality assessment. Clin chem. 1997; 43: 1114-1128.

9. Tully L A, Parsons T J, Steighner R J, Holland MM, Marino MA, Prenger VL, et al. A sensitive denaturing gradient Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region. Am J Hum Genet. 2000; 67: 432- 443.

10. Leonov SN, Zemskova EI, Timoshenko TV, Ivanov PL. The evaluation of the 287prospects for the application of mass-spectrometric analysis of the amplified 288 DNA fragments for the purpose of forensic medical expertise. Sud Med Ekspert. 2014; 57: 24–27.