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Supplemental material, sj-docx-11-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-2-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-13-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-12-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-17-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-18-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-19-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-16-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-9-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-12-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-17-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-3-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-8-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-14-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-8-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-15-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-18-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-19-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-11-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-1-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-9-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-2-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-1-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-15-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-4-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-16-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-docx-10-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Supplemental material, sj-pdf-3-msj-10.1177_13524585221097296 for Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis by Synne Brune, Einar A Høgestøl, Sigrid A de Rodez Benavent, Pål Berg-Hansen, Mona K Beyer, Ingvild Sørum Leikfoss, Steffan D Bos, Piotr Sowa, Cathrine Brunborg, Magi Andorra, Irene Pulido Valdeolivas, Susanna Asseyer, Alexander Brandt, Claudia Chien, Michael Scheel, Kaj Blennow, Henrik Zetterberg, Nicole Kerlero de Rosbo, Friedemann Paul, Antonio Uccelli, Pablo Villoslada, Tone Berge and Hanne F Harbo in Multiple Sclerosis Journal

Multiple sclerosis (MS) is an autoimmune, neurological disease. We investigated genome-wide DNA methylation profiles of CD4+ and CD8+ T cells from MS patients and healthy controls at baseline and a follow-up visit. Patients were all treatment-naïve at baseline, and either on treatment or remained untreated at the follow-up visit. MS patients show more changes in their T cell DNA methylation profiles as compared to healthy controls over time, with the most pronounced differences observed in the untreated MS patients. These findings underline the potential of DNA methylation as biomarkers in MS.

For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near CLEC16A at chromosome 16p13.13, encompassing among others CIITA, DEXI and SOCS1 in addition to CLEC16A. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in CLEC16A act as expression quantitative trait loci for CLEC16A itself in human pancreatic β-cells, for DEXI and SOCS1 in thymic tissue samples, and for DEXI in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the CIITA-DEXI-CLEC16A-SOCS1 gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of SOCS1 and CLEC16A in CD4+ T cells in samples homozygous for the risk allele of CLEC16A rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of CIITA, DEXI, CLEC16A and SOCS1. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in CLEC16A.

Master i biomedisin Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder of the central nervous system (CNS). Although, the cause of MS is still unknown, some genetical and environmental risk factor are known to be involved in the progression of MS. One of the environmental risk factors is low levels of vitamin D in serum. Vitamin D has many biological effects, and its action is mediated through its receptor vitamin D receptor (VDR, a nuclear receptor). This complex associates with retinoid X receptor (RXR), by which together will recognize specific DNA sequences, i.e. vitamin D response elements (VDREs), causing the regulation of genes that encode proteins involved in cell proliferation and differentiation of immune cells. CD8+ T cells, is likely to play a role in MS pathogenesis. More than 200 single nucleotide polymorphisms (SNPs) are identified to be associated with MS. We wanted therefore, to analyse if these MS risk variants can influence VDR binding site. But before we do so, the technique used to study binding of VDR to DNA (VDR chromatin immunoprecipitation) as well as activation of CD8+ T cell procedures must be optimized. CD8+ T cells were isolated from blood from healthy donors and were activated using 5 μg/ml anti-CD3 coated plate and 2 μg/ml soluble anti-CD28 antibodies for 40 hours to induce the expression of VDR. After 40 hours, VDR was highly expressed, and the cells were treated with either active form of vitamin D (calcitriol) or EtOH (vehicle control) for 3 hours. To verify the cells responsiveness to vitamin D, the expression of vitamin D responsiveness genes, TAGAP and CYP24A1 were measured. Furthermore, the ChIP procedure that includes (before immunoprecipitation) protein-DNA crosslinking (incubation temperature and time), cell lysis (one step or two step) and sonication to fragment chromatin (fragmentating for 10 or 20 min) was optimized prior to be performed in two healthy donors using anti-VDR to pull down DNA regions that are bound by VDR. Anti-IgG (isotype control) immunoprecipitation was performed as negative control. PCR of regions in the VDR, MYC and TAGAP was used to analyse the DNA fragments purified after ChIP. DNA regions that were pulled down by IgG were the same as VDR. So, given that our findings of ChIP are based on a limited number of experiments, the findings should be confirmed yet again doing more experiments. Therefore, immunoprecipitation part of the ChIP procedure needs to be optimized by including more materials and controls.