Allelic alterations of STRs in archival paraffin embedded tissue as DNA source for paternity testing
Received 6 August 2009; accepted 7 August 2009. published online 23 September 2009.
Abstract
Owing to a wrong name registered on ID card, the identity of a businessman who had been dead and cremated was suspected, which led his son failed to get legacy. In order to prove the parenthood, the son submitted the gastric cancer tissues surgically removed and embedded in a paraffin block as DNA source for paternity test. After extracting DNA with QIAamp DNA Blood Mini Kit, the 16 STR loci was amplified by two commercial kits of Sinofiler® (ABI)and Powerplex 16 (Promega), respectively. Both of the STR profiles were similarly showing allelic imbalance pattern at some loci and an additional allele at locus D18S51. The cancerous tissues and adjacent normal tissues were then partitioned off from each other by microscopic analysis of H.E. stained sections and followed by DNA extracting and STR typing, respectively. The allelic alteration could not be found in normal tissues whereas it did in cancerous tissues whose STR profile showed complete loss of one allele (LOH) at loci D13S317 (allele 11 was lost), partial loss of one allele (pLOH) at loci D21S11, D7S820, D19S433, vWA, D12S391 and Amelogein and occurrence of an additional allele (allele 20 was added) at locus D18S51. The results demonstrated that the Paraffin Embedded cancer Tissue used as DNA source for forensic identification is possibly questionable because of their microsatellite instability (MSI) or loss of heterozygosity. It was suggested to partition the normal tissues from the cancer tissues by microscopic evaluation first and then analyzing DNA separately. Comparing the STRs profile of normal tissue with the son's blood sample, the final conclusion was acquired that the donor of the paraffin embedded tissues is the biological father of the son.
An overseas businessman, who had invested a food corporation in a south city of china, unfortunately died of stomach cancer in a hospital. According to law, his son could inherit all of his possessions. But during the process of transacting relevant business, the son was encountered a trouble that the dead man's name on his overseas ID card was inconsistent with what was registered in mainland. In order to testify himself as a heir at law, the son asked to determine the identity of the businessman by means of paternity testing. Because the businessman's body was cremated and the only remains of him was surgically removed gastric tissues embedded in paraffin block saved by a hospital, as the source of DNA, both the paraffin embedded tissue and the son's blood samples were delivered to DNA lab for the purpose of paternity test.
1. Materials and methods
1.1. Sample pareparation
The paraffin embedded tissue block was sliced into three 5μm sections with microtome. One of the sections was directly put in a 1.5mL micro centrifuge tube tagged “sample P.E-both” and removed wax off in xylene at least for 45min. The other two sections were mounted on slide and stained with haematoxylin eosin (H.E) and distinguished non-cancerous and cancerous tissue by microscopic examination, then the two microslides were immerged in xylene until the coverslips were fallen off and then dried in the air. On one of the slides, the cancerous tissue and adjacent non-cancerous tissue were in caution respectively transferred to two 1.5mL micro centrifuge tubes tagged “sample H.E-Can.” and “sample H.E-Nor”. As contrast, the overall stained tissues on another slide were transferred to a 1.5mL micro centrifuge tube tagged “sample H.E-both”. Finally, all of these four prepared samples were washed by 100% ethanol for two times and dried on 40°C heater block for 1h.
1.2. DNA extraction, quantification and PCR
DNA from all above prepared samples were extracted using QIAamp DNA Blood Mini Kit according to the manufacturer's instructions and the final volume of eluted DNA solution was 50μL. DNA of blood sample was extracted by Chelex-100 method. The extracted DNA were assessed by Real-Time PCR using Human Quantifiler™ kit (ABI). For genotyping, the Sinofiler® (ABI) and Powerplex 16 (Promega) kits were used and polymerase chain reaction (PCR) was performed taking approximately 2ng DNA in a total volume of 12.5μL. Amplification was carry out using a GeneAmp PCR-System 9700 (PerkinElmer Applied Biosystem). The amplification product was separated and analyzed using an ABI PRISM 3100 Genetic Analyzer.
1.3. Genetic analysis of paternity
The results were expressed as the parenthood likelihood ratio of the donor of non-cancerous tissues versus the son, which was caculated upon the datas of STR allele frequencies from China Han population
2. Results and discussions
As a kind of archival biological material, paraffin embedded tissue sometimes is only DNA source for forensic identification or paternity test. However, most paraffin embedded tissues, especially surgically removed and saved in hospital, were pathologic tissues from patients. It had been demonstrated that cancer specimens showed great variability in their genetic phenotypes due to microsatellite instability (MSI) or loss of heterozygosity (LOH)[1], [2], [3], [4], which possibly resulted in a false interpretation in forensic casework. In the present case, the whole sliced paraffin embedded tissue section (“sample P.E-both”) was first routinely analyzed by DNA typing and it was noticed that the heterozygote imbalance (i.e., peak height of one allele is reduced to less than 0.80) at multiple loci D7S820, D13S317, vWA and Amelogenin and an additional allele at locus D18S51 in STR profile. When using another commercial kit-PowerPlex 16 for analyzing, the similar STR profile showing allelic alteration was acquired. In order to testify the real incidence of the allelic alterations caused by microsatellite instability (MSI) of tumour tissue, the H.E. stained non-cancerous and cancerous tissue fractions on the microscopic slides were distinguished by pathologist and were possessed DNA extracting and STR profiling, respectively. The STR profile of non-cancerous tissue showed no allelic alteration (Fig. 1a) whereas cancerous tissue showed allelic alteration including complete loss of one allele (LOH) at loci D13S317 (allele 11 was lost), partial loss of one allele (pLOH) at loci D21S11, D7S820, D19S433, vWA, D12S391 and Amelogein, occurrence of an additional allele (allele 20 was added) at locus D18S51 (Fig. 1b). The same with the unstained paraffin embedded tissue section (“sample P.E-both”), the allelic imbalance pattern of STRs profile was obtained in sample H.E.-both (Fig. 1c).
Fig. 1. The STR profile of H.E. stained sections which non-cancerous tissue (a), cancerous tissue (b) and both (c).
Kinship analysis was subsequently processed by comparing data of allele from non-cancerous tissues with the son's blood sample. It was clear that the obligate gene of the son could be found in the source of the paraffin embedded tissues. The combined paternity index (PI:X/Y) was 206438.36. It was finally supported that the contributor of the paraffin embedded tissue was the biological father of the son.
In process of paternity testing, when using archival paraffin embedded tumor tissue specimens as DNA source, it had a potential risk of false homozygosity or heterozygosity because of the allele deletion or addition, which would finally result in false conclusion especially in the situation when the alleles lost or altered happened to be the alleles that transmitted by parents to children. Therefore, when the allelic imbalance pattern on STR profile from archived paraffin embedded tumor tissues was encountered, the MSI and LOH should be interpreted carefully. The best strategy was to partition cancerous tissues and adjacent normal tissues from each other after microscopic evaluation with H.E. staining, and followed by DNA extracting and STR typing, respectively.
Conflict of interest
None.
Acknowledgements
Special thanks are given to Kaifei Deng for his excellent preparation of H.E. stained paraffin embedded tissue sections and also to professor Ningguo liu for his expert pathology evaluation by microscope.
References
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Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Sciences, Ministry of Justice, 1347 West Guangfu Road, Shanghai 200063, PR China
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