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Bone marrow chimerism detection using next generation sequencing based on single nucleotide polymorphisms following liver transplantation: comparison with short tandem repeat-PCR.

Kim J, Hwang IS, Kim HS, Joo DJ, Hong KR, Choi JR - Ann Lab Med (2016)

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine, Yonsei University College of Medicine, Seoul, Korea.

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Dear Editor, Bone marrow chimerism (BMC) develops after allogeneic hematopoietic stem cell transplantion (HSCT); however, BMC is also associated with graft-versus-host disease (GVHD) following liver transplantation, which has an incidence rate of 0.1-2% and a mortality rate exceeding 75%... The percentage of recipient DNA determined in mixed chimerism analyses were 5.6% (range 4.1-7.7%) by STR-PCR and 4.9% (range 2.5-6.7%) by NGS... The percent recipient DNA determined by NGS was equivalent to that determined by STR-PCR, indicating its applicability in chimerism analysis... Pancytopenia after SOT is the presenting symptom of life-threatening pathologies, such as SOT-GVHD, post-transplant lymphoproliferative disorder, or unsuspected opportunistic infection... Although lymphocyte macrochimerism can occur transiently and decrease rapidly over time, usually within the third postoperative week, this study illustrates an occurrence of BMC six weeks after liver transplantation, with 95.1% of the DNA originating from the donor... This high donor chimerism has been associated with liver transplants from deceased donors and recipients with hepatitis C virus... The relative amount of DNA from the recipient was 4.9%, which was too low to be reliably distinguished from background noise and could cause misinterpretation in cases of mixed chimerism... Here, we used NGS to quantify chimerism and confirmed the applicability of this method by comparing with conventional STR-PCR... NGS is an emerging, promising diagnostic tool for a number of clinical applications and forensic analyses... The present observations may have implications in the field of transplantation research and exhibit the diagnostic utility of this new technology.

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NGS chimerism analysis 41 days after liver transplant. Thirty-two of ninety SNPs discriminated between the donor and recipient. The average percent of donor DNA was 95.1%. Ninety SNPs are shown for (A) the recipient (peripheral blood, pre-transplant), (B) donor (peripheral blood), and (C) recipient (bone marrow, post-transplant) with mean coverage depth for each SNPs, 674, 1,609 and 1,080, respectively. The following markers were used for the chimerism calculation: rs560681, rs891700, rs12997453, rs6444724, rs2046361, rs159606, rs251934, rs338882, rs13218440, rs727811, rs10092491, rs2056277, rs1463729, rs735155, rs740598, rs964681, rs2076848, rs2269355, rs2111980, rs1058083, rs354439, rs873196, rs2016276, rs1382387, rs9905977, rs2292972, rs1736442, rs719366, rs1005533, rs722098, rs2830795, and rs987640.*discriminating SNPs used in chimerism calculation.Abbreviations: NGS, next generation sequencing; SNPs, single nucleotide polymorphisms.
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Figure 1: NGS chimerism analysis 41 days after liver transplant. Thirty-two of ninety SNPs discriminated between the donor and recipient. The average percent of donor DNA was 95.1%. Ninety SNPs are shown for (A) the recipient (peripheral blood, pre-transplant), (B) donor (peripheral blood), and (C) recipient (bone marrow, post-transplant) with mean coverage depth for each SNPs, 674, 1,609 and 1,080, respectively. The following markers were used for the chimerism calculation: rs560681, rs891700, rs12997453, rs6444724, rs2046361, rs159606, rs251934, rs338882, rs13218440, rs727811, rs10092491, rs2056277, rs1463729, rs735155, rs740598, rs964681, rs2076848, rs2269355, rs2111980, rs1058083, rs354439, rs873196, rs2016276, rs1382387, rs9905977, rs2292972, rs1736442, rs719366, rs1005533, rs722098, rs2830795, and rs987640.*discriminating SNPs used in chimerism calculation.Abbreviations: NGS, next generation sequencing; SNPs, single nucleotide polymorphisms.

Mentions: BMC was also analyzed with NGS by using the HID-Ion AmliSeq Identity Panel (Life Technologies, Milan, Italy). Bar-coded libraries were generated by using the Ion Xpress Plus Fragment Library Kit (Life Technologies) and Ion Xpress DNA Bar Coding Kit (Life Technologies) with adaptors. The panel selectively analyzed gene content by discriminating 120 SNPs, including 90 autosomal SNPs and 30 upper Y-Clade SNPs. Since both the donor and recipient were women, 30 upper Y-Clade SNPs were not used. Out of 90 SNPs, 32 sequence variations were observed and read number of variant sequence per read number of expected sequence was calculated to determine percent chimerism (Fig. 1).


Bone marrow chimerism detection using next generation sequencing based on single nucleotide polymorphisms following liver transplantation: comparison with short tandem repeat-PCR.

Kim J, Hwang IS, Kim HS, Joo DJ, Hong KR, Choi JR - Ann Lab Med (2016)

NGS chimerism analysis 41 days after liver transplant. Thirty-two of ninety SNPs discriminated between the donor and recipient. The average percent of donor DNA was 95.1%. Ninety SNPs are shown for (A) the recipient (peripheral blood, pre-transplant), (B) donor (peripheral blood), and (C) recipient (bone marrow, post-transplant) with mean coverage depth for each SNPs, 674, 1,609 and 1,080, respectively. The following markers were used for the chimerism calculation: rs560681, rs891700, rs12997453, rs6444724, rs2046361, rs159606, rs251934, rs338882, rs13218440, rs727811, rs10092491, rs2056277, rs1463729, rs735155, rs740598, rs964681, rs2076848, rs2269355, rs2111980, rs1058083, rs354439, rs873196, rs2016276, rs1382387, rs9905977, rs2292972, rs1736442, rs719366, rs1005533, rs722098, rs2830795, and rs987640.*discriminating SNPs used in chimerism calculation.Abbreviations: NGS, next generation sequencing; SNPs, single nucleotide polymorphisms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697352&req=5

Figure 1: NGS chimerism analysis 41 days after liver transplant. Thirty-two of ninety SNPs discriminated between the donor and recipient. The average percent of donor DNA was 95.1%. Ninety SNPs are shown for (A) the recipient (peripheral blood, pre-transplant), (B) donor (peripheral blood), and (C) recipient (bone marrow, post-transplant) with mean coverage depth for each SNPs, 674, 1,609 and 1,080, respectively. The following markers were used for the chimerism calculation: rs560681, rs891700, rs12997453, rs6444724, rs2046361, rs159606, rs251934, rs338882, rs13218440, rs727811, rs10092491, rs2056277, rs1463729, rs735155, rs740598, rs964681, rs2076848, rs2269355, rs2111980, rs1058083, rs354439, rs873196, rs2016276, rs1382387, rs9905977, rs2292972, rs1736442, rs719366, rs1005533, rs722098, rs2830795, and rs987640.*discriminating SNPs used in chimerism calculation.Abbreviations: NGS, next generation sequencing; SNPs, single nucleotide polymorphisms.
Mentions: BMC was also analyzed with NGS by using the HID-Ion AmliSeq Identity Panel (Life Technologies, Milan, Italy). Bar-coded libraries were generated by using the Ion Xpress Plus Fragment Library Kit (Life Technologies) and Ion Xpress DNA Bar Coding Kit (Life Technologies) with adaptors. The panel selectively analyzed gene content by discriminating 120 SNPs, including 90 autosomal SNPs and 30 upper Y-Clade SNPs. Since both the donor and recipient were women, 30 upper Y-Clade SNPs were not used. Out of 90 SNPs, 32 sequence variations were observed and read number of variant sequence per read number of expected sequence was calculated to determine percent chimerism (Fig. 1).

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine, Yonsei University College of Medicine, Seoul, Korea.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Dear Editor, Bone marrow chimerism (BMC) develops after allogeneic hematopoietic stem cell transplantion (HSCT); however, BMC is also associated with graft-versus-host disease (GVHD) following liver transplantation, which has an incidence rate of 0.1-2% and a mortality rate exceeding 75%... The percentage of recipient DNA determined in mixed chimerism analyses were 5.6% (range 4.1-7.7%) by STR-PCR and 4.9% (range 2.5-6.7%) by NGS... The percent recipient DNA determined by NGS was equivalent to that determined by STR-PCR, indicating its applicability in chimerism analysis... Pancytopenia after SOT is the presenting symptom of life-threatening pathologies, such as SOT-GVHD, post-transplant lymphoproliferative disorder, or unsuspected opportunistic infection... Although lymphocyte macrochimerism can occur transiently and decrease rapidly over time, usually within the third postoperative week, this study illustrates an occurrence of BMC six weeks after liver transplantation, with 95.1% of the DNA originating from the donor... This high donor chimerism has been associated with liver transplants from deceased donors and recipients with hepatitis C virus... The relative amount of DNA from the recipient was 4.9%, which was too low to be reliably distinguished from background noise and could cause misinterpretation in cases of mixed chimerism... Here, we used NGS to quantify chimerism and confirmed the applicability of this method by comparing with conventional STR-PCR... NGS is an emerging, promising diagnostic tool for a number of clinical applications and forensic analyses... The present observations may have implications in the field of transplantation research and exhibit the diagnostic utility of this new technology.

No MeSH data available.