A case of false mother included with 46 autosomal STR markers

Background For solving a maternity case, 19 autosomal short tandem repeats (STRs) were amplified using the AmpFℓSTR® SinofilerTM kit and PowerPlex® 16 System. Additional 27 autosomal STR loci were analyzed using two domestic kits AGCU 21+1 and STRtyper-10G. The combined maternity index (CMI) was calculated to be 3.3 × 1013, but the putative mother denied that she had given birth to the child. In order to reach an accurate conclusion, further testing of 20 X-chromosomal short tandem repeats (X-STRs), 40 single nucleotide polymorphism (SNP) loci, and mitochondrial DNA (mtDNA) was carried out. Findings The putative mother and the boy shared at least one allele at all 46 tested autosomal STR loci. But, according to the profile data of 20 X-STR and 40 SNP markers, different genotypes at 13 X-STR loci and five SNP loci excluded maternity. Mitochondrial profiles also clearly excluded the mother as a parent of the son because they have multiple differences. It was finally found that the putative mother is the sister of the biological father. Conclusions Different kinds of genetic markers needfully supplement the use of autosomal STR loci in case where the putative parent is suspected to be related to the true parent.


Background
Profiles derived from polymorphic short tandem repeats (STRs) are used worldwide in paternity testing and individual identification. In complex cases of kinship analysis, autosomal single nucleotide polymorphism (SNP), X-chromosomal short tandem repeat (X-STRs), and mitochondrial DNA (mtDNA) could be used to complement autosomal STR typing.
Genotypes of STRs and X-STRs are routinely determined using commercial PCR-based amplification kits with subsequent fragment length determination using capillary electrophoresis with laser-induced fluorescence of labeled primers.
Several years ago, SNP and mtDNA analysis using a PCR and electrospray ionization mass spectrometrybased methods [1,2] have been developed and reported. For a given individual, the PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS)-based assay provides a simple profile consisting of a read-out of 40 binary autosomal SNP markers identified by the Kidd laboratory in 2007 [3]. Meanwhile, this assay offers an efficient high throughput method for profiling the control region of mtDNA that identifies differences between individuals without targeting specific nucleotide positions. This approach provides resolution exceeding that obtained by sequencing the minimum HV1 and HV2 coordinates (16024-16365 and 73-340) by determining the base compositions of 24 short (80-120 bp) amplicons derived from tiling primers covering coordinates 15924-16428 and 31-576 [4,5].
In this paper we describe an interesting case. A couple went to the Public Security Bureau to declare account for a boy, they claimed the child was abandoned two years ago shortly after birth, and they picked him up and brought him up to date. In order to prove that the boy was really not their child, parentage testing was performed as officially requested. The putative mother (M) shared at least an allele with the boy (B) at each of the 46 autosomal STRs detected, but the putative father was excluded as the biological father with 18 inconsistent loci out of the 46 markers. The mother was adamant that she had not given birth to the child. Below, we emphatically describe the use of additional non-STR genetic markers (SNPs, X-STRs, and mtDNA) to interrogate the potential maternal association between the putative mother and child.

Methods
Blood samples from the putative mother and the child were collected with informed consent under protocols approved by the IFS ethics committee at the Institute of Forensic Science, Ministry of Justice, China. DNA extraction was performed using a Chelex-100 and proteinase K protocol [6]. The quantity of DNA derived was determined spectrophotometrically and was subsequently aliquoted into the various kits following manufacturer's guidelines.
Capillary electrophoresis-based STR and X-STR typing was performed on an Applied Biosystems 3130XL genetic analyzer following PCR on an Applied Biosystems GeneAmp 9700 thermalcycler. Data was analyzed using Applied Biosystem's GeneMapper software V3.2. Commercial autosomal STR panels employed in this work include Applied Biosystem's 16-marker AmpFℓSTR Sinofiler and Promega's 16-marker PowerPlex 16. Twenty seven additional autosomal STRs were interrogated using the domestic 21+1 kit [7] and the Typer 10 panel [8]. The commercial X-chromosomal STR panel employed in this work was Mentype® Argus X-8 Kit (Biotype® AG, Germany) [9]. Additional X-chromosomal STRs were interrogated using the in-house IDtyper X-16 kit [10].
Mass spectrometry-based SNP and mtDNA typing was performed using Ibis Biosciences' PLEX-ID platform [11]. A set of primer pairs to amplify 40 autosomal SNP loci were arranged into a panel of eight 5-plex reactions. Twenty-four primer pairs in eight 3-plex reactions were employed to tile across an extended HV1/HV2 domain of the mitochondrial genome corresponding to coordinates 15924-16428 and 31-576. Genotypes of SNP markers and base compositions (i.e., the number of A's, G's, C's, and T's) of each amplicon of mtDNA were determined using fully automated high throughput mass spectrometry on PLEX-ID platforms.

STR analysis
As shown in Table 1, the putative father was clearly excluded as being the biological father because there was 18 inconsistent loci out of the 46 tested autosomal STRs. However, at each of the 46 loci, there was at least one shared allele between the putative mother and the child  (Table 1). Based on the allele frequencies of Chinese Han population, the combined maternity index (CMI) of 3.3 × 10 13 by no means excludes the putative mother from being the biological mother. The putative mother refused the conclusion that she was the real mother. In order to get at the facts, the likelihoods of the genotype profiles given various identity-bydescent (IBD) distributions were then calculated by J Ge and B Budowle using MPKin [12,13]. According to the analysis results, paternal aunt nephew relationship was very likely. Table 2 depicts the X-STR profiles of the putative mother and the boy at the 20 X-chromosomal STR loci interrogated by capillary electrophoresis. The putative mother was clearly excluded as being the biological mother as her X-STR profile was not consistent with that of the child at 13 out of the 20 X-STR loci detected.

Autosomal SNPs analysis
In this study, the full 40 SNP panel was run on DNA derived from the putative mother and the child. As illustrated in Table 3, there are five independent loci with opposite homozygous genotypes. Because the average mutation frequency is 1 in 10 6 for a given SNP locus, there would be approximately a 1 in a million probability for a child to have an allele that is inconsistent with the mother's genotype at a single locus and less than a 1 in 10 30 probability of having five loci inconsistent with the biological mother's genotype. These data clearly exclude the putative mother from being the biological mother of the child.

Mitochondrial DNA analysis
In this study, mitochondrial profiles were derived from the putative mother and child and compared. As illustrated in Table 4, there are clear and obvious differences between the two profiles, further corroborating the exclusion suggested by the X-STR and SNP data above. It is apparent that the child has considerable C-length heteroplasmy in HV1 and HV2 which is not apparent in the profile of the putative mother. For example, for primer pair 2896 which covers coordinates 16102..16224, a single base composition of A45 G13 C41 T24 is observed while the same primer pair yields multiple length variants of these coordinates spanning four C-length variants with base compositions of A44 G13 C43 T22 to A44 G13 C46 T22. Note also that the putative mother has 45 A's and 24 T's over these coordinates and the child, regardless of C-length variation, consistently has 44 A's and 22 T's. These differing base compositions  represent multiple clear and unique differences between the mitochondrial profiles of the child and the putative mother over the coordinates spanned by a single primer pair. Perhaps more importantly, there are clear and distinct base composition differences in 9 of the 24 primer pairs, clearly and unambiguously inconsistent with a profile shared between child and biological mother.

Discussions and conclusions
Forty-six autosomal STRs, 20 X-STRs, 40 SNPs, and mtDNA were typed for the resolved case. Calculated on the basis of population genetics data [7][8][9][10]14], in Chinese Han population, the accumulative exclusion power of the 46 autosomal loci and 20 X-STR markers in duos was 0.999999999999986 and 0.999999948, respectively.