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Donor-derived DNA variability in fingernails of acute myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation detected by direct PCR

Human fingernails are an important material used to identify individuals in forensic medicine as well as a source of DNA for genetic analyses in different fields of study. Nail clippings have many advantages, such as stability and suitability for long-term storage, easy non-invasive sampling and simple transport [1, 2]. On the other hand, the structure of a fingernail (presence of keratinized cells) requires a more complicated DNA extraction method than DNA extraction from blood cells. Therefore, it is advantageous to use a direct PCR method that does not require a DNA isolation step. This method was used on fingernails for the first time by Tie et al. [3], and by Ottens et al. [4]. The benefits of the method are simplicity, reducing the possibility of contamination and time- and cost-saving. We used this innovative method for donor-derived DNA analysis on the fingernails of 30 acute myeloid patients (AML) with long-lasting complete donor chimerism in their peripheral blood after the first allogeneic hematopoietic stem cell transplantation (allo-HSCT).

The nails were collected between 217 to 6169 days after the first allo-HSCT from patients that had complete donor chimerism (CC) in the peripheral blood at least 110 days before nail collection. Specific short tandem repeat (STR) polymorphisms were determined for each donor-recipient pair in the peripheral blood (before allo-HSCT) and the same STR polymorphisms were used for fingernails analysis by direct PCR. A total of 15 different STR markers were used for fingernail analysis. In 26/30 cases we were able to amplify at least two informative STR polymorphisms per patient by direct PCR. Each fingernail (at least one per patient) was divided into 3–6 fragments and each of these fragments was analyzed by direct PCR in two ways: (i) as a pooled sample (in an equivalent ratio) composed from the same patient’s partial nail fragments to analyze interindividual variability, and (ii) as separate samples of partial nail fragments to identify intraindividual variability.

Analyses of nails obtained from different individuals after allo-HSCT showed that donor-derived DNA is present in all amplifiable fingernails and next to the DNA from recipient cells. In addition, the proportion of interindividual variability had a wide range from 4 to 95% and did not correspond to the time from the first allo-HSCT (R2 = 0.0030) or complete donor chimerism (CC) duration (R2 = 0.0009). Similarly, interindividual variability in fingernails has been detected by Sanz-Pina et al. [5] using non-direct PCR in a cohort of 20 transplanted patients. However, most of the fingernail samples (13/20) were collected within 4 months of transplantation [5]. Regarding nail growth, the clipped fingernails reflect the nail matrix status ~5–6 months before clipping [6]. Also Imanishi et al. [7] identified donor-derived DNA presence in the fingernails (collected between 305 and 2399 days from allo-HSCT) of 9/21 patients by non-direct PCR [7]. Although the presence of DNA from donor cells in patients’ fingernails after allo-HSCT appears well documented, the mechanism of this phenomenon remains unclear. In other experimental and clinical studies, the presence of donor derived DNA was also identified in other nonhematological patient tissue after allo-HSCT, e.g., in tissue of lung [8], liver [9], skeletal and cardiac muscles [10, 11], or buccal swabs [12, 13]. Possible mechanisms to explain the epithelial chimerism phenomenon are (donor) hematopoietic stem cell conversion into epithelial cells or spontaneous somatic cell fusion with embryonic stem cells [14, 15]. Recently, Waterhouse et al. [13] described another alternative mechanism. They hypothesize that the possible operating mechanism can be a horizontal hematopoietic donor DNA transfer to the host epithelium. They assume that after allo-HSCT, there is a continuous transfer and integration of donor genomic material from apoptotic cells into the host epithelium that results in emerging epithelial cells that contain donor-derived DNA [13]. They validated this assumption by detecting a high amount of donor-derived DNA in transplant recipient buccal swabs, where no donor hematopoietic cells were detected by immunofluorescence [13].

Our other analyses focused on detecting donor-derived DNA in different fragments of the same patient´s fingernail by direct PCR. Unexpectedly, we revealed that various parts of the same fingernail showed different levels of donor-derived DNA ranging from negativity to almost full donor DNA positivity. We observed this phenomenon in all 26 analyzed patients’ fingernails. In standard non-direct PCR protocol, the whole nail clipping is usually used for DNA isolation. The obtained results of the analysis thus reflect the mean value of the whole nail. Direct PCR allowed us to obtain the results for individual small parts (segments) from a single fingernail. To our knowledge, the phenomenon of the inhomogeneous donor-derived DNA occurrence in the transplant recipient fingernail has not been described before and is an obvious benefit of the direct PCR method used. However, the mechanism of this event remains unclear. We assume a random event based on one of the mechanisms discussed above, together with varying intensity in different parts of the nail matrix, which is subsequently (after some time) detectable in the nail plate.

Our analyses showed that the direct PCR method is a widely applicable, simple and effective method to analyze DNA in fingernails. This method allowed us to observe the interindividual variability and unexpected intraindividual variability of inhomogeneous donor-derived DNA occurrence in AML patients’ fingernails with long-lasting complete donor chimerism in the peripheral blood after allo-HSCT. Our results can contribute to epithelial chimerism mechanism studies in patients after allo-HSCT.

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Acknowledgements

Supported by Ministry of Health, Czech Republic—conceptual development of research organization (FNBr, 65269705).

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BR, JM, and IJ designed the study. BR, MD, and AF performed experiments. BR, AF, and IJ analyzed data. MK, FF, and ZS provided the patients samples and clinical data. BR, AF, and IJ wrote the article. MD, MK, FF, ZS, and JM revised the manuscript. All authors have read and agreed with the final version of the manuscript.

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Correspondence to Ivana Jeziskova.

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Robesova, B., Drncova, M., Folta, A. et al. Donor-derived DNA variability in fingernails of acute myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation detected by direct PCR. Bone Marrow Transplant 55, 1021–1022 (2020). https://doi.org/10.1038/s41409-020-0938-x

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