Unveiling APOL1 haplotypes in a predominantly African-American cohort of kidney transplant patients: a novel classification using probe-independent quantitative real-time PCR.

Introduction: Apolipoprotein-L1 (APOL1) is a primate-specific protein component of high-density lipoprotein (HDL). Two variants of APOL1 (G1 and G2), provide resistance to parasitic infections in African Americans but are also implicated in kidney-related diseases and transplant outcomes in recipients. This study aims to identify these risk variants using a novel probe-independent quantitative real-time PCR method in a high African American recipient cohort. Additionally, it aims to develop a new stratification approach based on a haplotype-centric model. Methods: Genomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. A quantitative PCR assay with modified forward primers and a common reverse primer enabled us to quantitatively identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion. Additionally, we used Sanger sequencing to verify our QPCR findings. Results: Our novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletions, and homozygous SNPs/deletions, with at least 4-fold differences. A high prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, no significant impact of recipient APOL1 variants on transplant outcomes was observed up to 12-month of follow-ups. Ongoing research will encompass more time points and a larger patient cohort, allowing for a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding. Conclusion: Our cost-effective and rapid qPCR technique facilitates APOL1 genotyping within hours. Prospective and retrospective studies will enable comparisons with long-term allograft rejection, potentially predicting early/late-stage transplant outcomes based on haplotype evaluation in this diverse group of kidney transplant recipients.

Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR.

Introduction: Apolipoprotein-L1 (APOL1) is a primate-specific protein component of high- density lipoprotein (HDL). Two variants of APOL1 (G1 and G2), provide resistance to parasitic infections in African Americans but are also implicated in kidney-related diseases and transplant outcomes in recipients. This study aims to identify these risk variants using a novel probe- independent quantitative real-time PCR method in a high African American recipient cohort. Additionally, it aims to develop a new stratification approach based on haplotype-centric model. Methods: Genomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. Quantitative PCR assay with modified forward primers and a common reverse primer enabled us to identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion quantitatively. Additionally, we used sanger sequencing to verify our QPCR findings. Results: Our novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletion, and homozygous SNPs/deletion, with at least 4-fold differences. High prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, up to 12-month follow-up revealed no significant impact of recipient APOL1 variants on transplant outcomes. Ongoing research will encompass more time points and a larger patient cohort, allowing a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding. Conclusions: Our cost-effective and rapid qPCR technique facilitates APOL1 genotyping within hours. Prospective and retrospective studies will enable comparisons with long-term allograft rejection, potentially predicting early/late-stage transplant outcomes based on haplotype evaluation in this diverse group of kidney transplant recipients.

Circulating microRNA Profiles for Premature Cardiovascular Death in Patients with Kidney Failure with Replacement Therapy.

INTRODUCTION: Patients with kidney failure with replacement therapy (KFRT) suffer from a disproportionately high cardiovascular disease burden. Circulating small non-coding RNAs (c-sncRNAs) have emerged as novel epigenetic regulators and are suggested as novel biomarkers and therapeutic targets for cardiovascular disease; however, little is known about the associations of c-sncRNAs with premature cardiovascular death in KFRT. METHODS: In a pilot case-control study of 50 hemodialysis patients who died of cardiovascular events as cases, and 50 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we performed c-sncRNAs profiles using next-generation sequencing to identify differentially expressed circulating microRNAs (c-miRNAs) between the plasma of cases and that of controls. mRNA target prediction and pathway enrichment analysis were performed to examine the functional relevance of differentially expressed c-miRNAs to cardiovascular pathophysiology. The association of differentially expressed c-miRNAs with cardiovascular mortality was examined using multivariable conditional logistic regression. RESULTS: The patient characteristics were similar between cases and controls, with a mean age of 63 years, 48% male, and 54% African American in both groups. We detected a total of 613 miRNAs in the plasma, among which five miRNAs (i.e., miR-129-1-5p, miR-500b-3p, miR-125b-1-3p, miR-3648-2-5p, and miR-3150b-3p) were identified to be differentially expressed between cases and controls with cut-offs of p < 0.05 and log2 fold-change (log2FC) > 1. When using more stringent cut-offs of p-adjusted < 0.05 and log2FC > 1, only miR-129-1-5p remained significantly differentially expressed, with higher levels of miR-129-1-5p in the cases than in the controls. The pathway enrichment analysis using predicted miR-129-1-5p mRNA targets demonstrated enrichment in adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy, and oxytocin signaling pathways. In parallel, the circulating miR-129-1-5p levels were significantly associated with the risk of cardiovascular death (adjusted OR [95% CI], 1.68 [1.01-2.81] for one increase in log-transformed miR-129-1-5p counts), independent of potential confounders. CONCLUSIONS: Circulating miR-129-1-5p may serve as a novel biomarker for premature cardiovascular death in KFRT.

Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient.

Operational tolerance (OT) after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. In this first-of-its-kind pilot study, we assessed the immune landscape associated with OT using single-cell analyses. Peripheral mononuclear cells from a kidney transplant recipient with OT (Tol), 2 healthy individuals (HC), and a kidney transplant recipient with normal kidney function on standard-of-care immunosuppression (SOC) were evaluated. The immune landscape of the Tol was drastically different from that of SOC and emerged closer to the profile of HC. TCL1A+ naive B cells and LSGAL1+ regulatory T cells (Tregs) were in higher proportions in Tol. We were unable to identify the Treg subcluster in SOC. The ligand-receptor analysis in HC and Tol identified interactions between B cells, and Tregs that enhance the proliferation and suppressive function of Tregs. SOC reported the highest proportion of activated B cells with more cells in the G2M phase. Our single-cell RNA sequencing study identified the mediators of tolerance; however, it emphasizes the requirement of similar investigations on a larger cohort to reaffirm the role of immune cells in tolerance.

Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis.

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

Key hepatoprotective roles of mitochondria in liver regeneration.

Treatment of advanced liver disease using surgical modalities is possible due to the liver's innate ability to regenerate following resection. Several key cellular events in the regenerative process converge at the mitochondria, implicating their crucial roles in liver regeneration. Mitochondria enable the regenerating liver to meet massive metabolic demands by coordinating energy production to drive cellular proliferative processes and vital homeostatic functions. Mitochondria are also involved in terminating the regenerative process by mediating apoptosis. Studies have shown that attenuation of mitochondrial activity results in delayed liver regeneration, and liver failure following resection is associated with mitochondrial dysfunction. Emerging mitochondria therapy (i.e., mitotherapy) strategies involve isolating healthy donor mitochondria for transplantation into diseased organs to promote regeneration. This review highlights mitochondria's inherent role in liver regeneration.

Mitochondrial Role in Oncogenesis and Potential Chemotherapeutic Strategy of Mitochondrial Infusion in Breast Cancer.

Triple negative breast cancer (TNBC) is one of the most aggressive cancers diagnosed amongst women with a high rate of treatment failure and a poor prognosis. Mitochondria have been found to be key players in oncogenesis and tumor progression by mechanisms such as altered metabolism, reactive oxygen species (ROS) production and evasion of apoptosis. Therefore, mitochondrial infusion is an area of interest for cancer treatment. Studies in vitro and in vivo demonstrate mitochondrial-mediated reduction in glycolysis, enhancement of oxidative phosphorylation (OXPHOS), reduction in proliferation, and an enhancement of apoptosis as effective anti-tumor therapies. This review focuses on mitochondrial dysregulation and infusion in malignancies, such as TNBC.

A Review of Defatting Strategies for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease is a huge cause of chronic liver failure around the world. This condition has become more prevalent as rates of metabolic syndrome, type 2 diabetes, and obesity have also escalated. The unfortunate outcome for many people is liver cirrhosis that warrants transplantation or being unable to receive a transplant since many livers are discarded due to high levels of steatosis. Over the past several years, however, a great deal of work has gone into understanding the pathophysiology of this disease as well as possible treatment options. This review summarizes various defatting strategies including in vitro use of pharmacologic agents, machine perfusion of extracted livers, and genomic approaches targeting specific proteins. The goal of the field is to reduce the number of necessary transplants and expand the pool of organs available for use.

MicroRNAs: Novel Targets in Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (IRI) is one of the main factors for early allograft dysfunction (EAD), which may lead to graft rejection, graft loss, or shortened graft life in liver transplantation. Hepatic IRI appears to be inevitable during the majority of liver procurement and transportation of donor organs, resulting in a cascade of biological changes. The activation of signaling pathways during IRI results in the up- and downregulation of genes and microRNAs (miRNAs). miRNAs are ~21 nucleotides in length and well-characterized for their role in gene regulations; they have recently been used for therapeutic approaches in addition to their role as biomarkers for many diseases. miRNAs that are associated with hepatic IRI in in vitro and in vivo animal models are comprehensively summarized in this review. In those studies, the manipulation of miRNAs has been shown for the inhibition of aggravated immune response, reduction of apoptosis, stimulation of tissue repair, and enhancement of cell recovery to attenuate liver damage. Therefore, the utilization of liver-specific miRNA holds great potential as a therapeutic agent to improve early allograft dysfunction, hepatic injury, and patient outcome.

Integrative Analyses of Circulating Small RNAs and Kidney Graft Transcriptome in Transplant Glomerulopathy.

Transplant glomerulopathy develops through multiple mechanisms, including donor-specific antibodies, T cells and innate immunity. This study investigates circulating small RNA profiles in serum samples of kidney transplant recipients with biopsy-proven transplant glomerulopathy. Among total small RNA population, miRNAs were the most abundant species in the serum of kidney transplant patients. In addition, fragments arising from mature tRNA and rRNA were detected. Most of the tRNA fragments were generated from 5' ends of mature tRNA and mainly from two parental tRNAs: tRNA-Gly and tRNA-Glu. Moreover, transplant patients with transplant glomerulopathy displayed a novel tRNA fragments signature. Gene expression analysis from allograft tissues demonstrated changes in canonical pathways related to immune activation such as iCos-iCosL signaling pathway in T helper cells, Th1 and Th2 activation pathway, and dendritic cell maturation. mRNA targets of down-regulated miRNAs such as miR-1224-5p, miR-4508, miR-320, miR-378a from serum were globally upregulated in tissue. Integration of serum miRNA profiles with tissue gene expression showed that changes in serum miRNAs support the role of T-cell mediated mechanisms in ongoing allograft injury.

Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.

Technical advancements in epigenomics and applications in transplantation.

PURPOSE OF REVIEW: To summarize recently developed next generation sequencing-based methods to study epigenomics and epitranscriptomics. To elucidate the potential applications of these recently developed methods in transplantation research. RECENT FINDINGS: There are several methods established with the collaborative efforts from different consortiums, such as ENCODE, Human Cell Atlas, and exRNA consortium to study role of epigenetics in human health. Rapid development in the sequencing technology also enabled the establishment of these genome-wide studies. This review specifically focuses on these techniques, such as EM-seq to study DNA methylation, CUT&RUN, and CUT&Tag to study histone/transcription factor--DNA interactions, ATAC-seq to study chromatin accessibility, Hi-C to explore 3D genome architecture and several methods to study epigenetics at single-cell level. In addition, we briefly mentioned recent efforts to study lncRNAs and extracellular miRNAs. SUMMARY: Technical advancements in genomics, particularly epigenomics, shed light on the role of epigenetics and recently epitranscriptomics in different fields. Application of those techniques to transplantation research is still very limited because of technical limitations. On the other hand, there are a lot of promising studies showing that these new techniques can be adapted to study the molecular biology of transplant-related problems.

FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are central in regulating immune responses of kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in mice treated with S1PR agonist, FTY720 (FTY). We tested if ex vivo propagation of DCs with FTY could be used as cellular therapy to limit the off-target effects associated with systemic FTY administration in kidney IRI. DCs have the ability of regulate innate and adaptive responses and we posited that treatment of DC with FTY may underlie improvements in kidney IRI. Herein, it was observed that treatment of bone marrow derived dendritic cells (BMDCs) with FTY induced mitochondrial biogenesis, FTY-treated BMDCs (FTY-DCs) showed significantly higher oxygen consumption rate and ATP production compared to vehicle treated BMDCs (Veh-DCs). Adoptive transfer of FTY-DCs to mice 24 h before or 4 h after IRI significantly protected the kidneys from injury compared to mice treated with Veh-DCs. Additionally, allogeneic adoptive transfer of C57BL/6J FTY-DCs into BALB/c mice equally protected the kidneys from IRI. FTY-DCs propagated from S1pr1-deficient DCs derived from CD11cCreS1pr1fl/fl mice as well as blunting mitochondrial oxidation in wildtype (WT) FTY-DCs prior to transfer abrogated the protection observed by FTY-DCs. We queried if DC mitochondrial content alters kidney responses after IRI, a novel but little studied phenomenon shown to be integral to regulation of the immune response. Transfer of mitochondria rich FTY-DCs protects kidneys from IRI as transferred FTY-DCs donated their mitochondria to recipient splenocytes (i.e., macrophages) and prior splenectomy abrogated this protection. Adoptive transfer of FTY-DCs either prior to or after ischemic injury protects kidneys from IRI demonstrating a potent role for donor DC-mitochondria in FTY's efficacy. This is the first evidence, to our knowledge, that DCs have the potential to protect against kidney injury by donating mitochondria to splenic macrophages to alter their bioenergetics thus making them anti-inflammatory. In conclusion, the results support that ex vivo FTY720-induction of the regulatory DC phenotype could have therapeutic relevance that can be preventively infused to reduce acute kidney injury.

Applications of CRISPR technologies in transplantation.

In transplantation, the ever-increasing number of an organ's demand and long-term graft dysfunction constitute some of the major problems. Therefore, alternative solutions to increase the quantity and quality of the organ supply for transplantation are desired. On this subject, revolutionary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology holds enormous potential for the scientific community with its expanding toolbox. In this minireview, we summarize the history and mechanism of CRISPR/Cas9 systems and explore its potential applications in cellular- and organ-level transplantation. The last part of this review includes future opportunities as well as the challenges in the transplantation field.

Long Noncoding RNA DRAIC Inhibits Prostate Cancer Progression by Interacting with IKK to Inhibit NF-κB Activation.

DRAIC is a 1.7 kb spliced long noncoding RNA downregulated in castration-resistant advanced prostate cancer. Decreased DRAIC expression predicts poor patient outcome in prostate and seven other cancers, while increased DRAIC represses growth of xenografted tumors. Here, we show that cancers with decreased DRAIC expression have increased NF-κB target gene expression. DRAIC downregulation increased cell invasion and soft agar colony formation; this was dependent on NF-κB activation. DRAIC interacted with subunits of the IκB kinase (IKK) complex to inhibit their interaction with each other, the phosphorylation of IκBα, and the activation of NF-κB. These functions of DRAIC mapped to the same fragment containing bases 701-905. Thus, DRAIC lncRNA inhibits prostate cancer progression through suppression of NF-κB activation by interfering with IKK activity. SIGNIFICANCE: A cytoplasmic tumor-suppressive lncRNA interacts with and inhibits a major kinase that activates an oncogenic transcription factor in prostate cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/5/950/F1.large.jpg.

Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3-mediated gene silencing.

tRNA fragments (tRFs) and tRNA halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more. Overexpressed angiogenin (ANG) cleaves tRNA anticodons and produces tRNA halves similar to those produced in response to stress. However, it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRFs are generated from the tRNA halves. Here, using global short RNA-Seq approach, we found that ANG overexpression selectively cleaves a subset of tRNAs, including tRNAGlu, tRNAGly, tRNALys, tRNAVal, tRNAHis, tRNAAsp, and tRNASeC to produce tRNA halves and tRF-5s that are 26-30 bases long. Surprisingly, ANG knockout revealed that the majority of stress-induced tRNA halves, except for the 5' half from tRNAHisGTG and the 3' half from tRNAAspGTC, are ANG independent, suggesting there are other RNases that produce tRNA halves. We also found that the 17-25 bases-long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from tRNA halves. Consistent with this, ANG knockout did not decrease tRF-3 levels or gene-silencing activity. We conclude that ANG cleaves specific tRNAs and is not the only RNase that creates tRNA halves and that the shorter tRFs are not generated from the tRNA halves or from independent tRNA cleavage by ANG.

tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner.

tRNA related RNA fragments (tRFs), also known as tRNA-derived RNAs (tdRNAs), are abundant small RNAs reported to be associated with Argonaute proteins, yet their function is unclear. We show that endogenous 18 nucleotide tRFs derived from the 3' ends of tRNAs (tRF-3) post-transcriptionally repress genes in HEK293T cells in culture. tRF-3 levels increase upon parental tRNA overexpression. This represses target genes with a sequence complementary to the tRF-3 in the 3' UTR. The tRF-3-mediated repression is Dicer-independent, Argonaute-dependent, and the targets are recognized by sequence complementarity. Furthermore, tRF-3:target mRNA pairs in the RNA induced silencing complex associate with GW182 proteins, known to repress translation and promote the degradation of target mRNAs. RNA-seq demonstrates that endogenous target genes are specifically decreased upon tRF-3 induction. Therefore, Dicer-independent tRF-3s, generated upon tRNA overexpression, repress genes post-transcriptionally through an Argonaute-GW182 containing RISC via sequence matches with target mRNAs.

Biogenesis and Function of Transfer RNA-Related Fragments (tRFs).

Noncoding small RNAs arise from the various un-annotated and annotated regions of the genome. When they arise from annotated genes, the noncoding small RNAs are functionally different from the parent genes. This is a brief review of one class of noncoding small RNAs, tRNA-related fragments (tRFs), which are generated from tRNA. tRFs have been suggested to have roles in cell proliferation, priming of viral reverse transcriptases, regulation of gene expression, RNA processing, modulation of the DNA damage response, tumor suppression, and neurodegeneration.

A Pro-metastatic tRNA Pathway.

tRNAs are best known as basic modules for global regulation of protein synthesis. Goodarzi et al. now show that two tRNAs upregulated in metastatic breast cancer cells enhance stability and translation of transcripts enriched with these codons, leading to specific increase in production of pro-metastatic proteins.

Pyrosequencing Analysis for Breast Cancer DNA Methylome.

Unraveling DNA methylation profile of tumor is important for the diagnosis and treatment of cancer patients. Because of the heterogeneity of clinical samples, it is very difficult to get methylation profile of only tumor cells. Laser capture Microdissection (LCM) is giving us a chance to isolate the DNA only from the tumor cells without any stroma cell's DNA contamination. Once we capture the breast tumor cells, we can isolate the genomic DNA which is followed by the bisulfite treatment in which unmethylated cytosines of the CG pairs are converted into uracil; however, methylated cytosine does not go into any chemical change during this reaction. Next, bisulfite treated DNA is used in the regular PCR reaction to get a single band PCR amplicon which will be used as a template for the pyrosequencing. Pyrosequencing is a powerful method to make a quantitative methylation analysis for each specific CG pair.