My Power Seminar
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History and Development
What are circulating nucleic acids (CNAs) ? CNAs refers to cell-free segments of DNA or RNA found in the blood and other body fluids like lymph , milk , urine etc.
What constitute the CNAs? CNAs includes DNA , RNA , miRNA.
Both DNA (1.8 - 35 ng mL-1) and RNA (2.5ng mL-1) are found in and plasma and serum from healthy donors.
Plasma
Where does the CNAs come from?
There are several possible sources of CNA, namely:
Break down of blood cellsBreak down of any pathogens, e.g. bacteria or virusesApoptosisNecrosisSpontaneous release of a newly synthesized DNASpontaneous release of DNA/RNA-lipoprotein complex from healthy cells
Random DNA fragmentat--ion which forms a "smear"
“ladder” pattern at ~200-b.p interval
Nucleosome -200 b.p
Linker region
DNA Fragmentation
Apoptosis
Necrosis
Apoptosis DNA V/S Necrosis DNA
800b.p 600b.p 400 b.p 200 b.p
~ 10000 b.p
CNAs in Cancer
Early detection of cancer can improve the success of treatment and reduce mortality rate .
In the past years the focus increases on studies of CNAs and cancer
Concentration of CNA
Cancer Concn. (ng/ml)
Ovarian >120
Prostate 450-749
Colorectal 105-709
Abnormalities of CNAs in Cancer : p-53 Mutation
K-RAS Mutation
Microsatellite Instability
Promoter Hypermethylation
Viral sequence e.g EBV , HPV
Rearranged Ig gene
p-53 Mutation
G:C -> A:T occurs at CpG dinucleotides at the codons 175, 248, 273 . e.g colon and breast cancer.
G->T occurs in lymphomas and leukemias . Due to mutation DNA binding capacity of p53
is affected.
p53 cannot carry out its normal function in cell cycle and DNA repair.
Tumour Develops
P53 is a tumour-suppressor gene
K-RAS – kirsten rat sarcoma virus homolog Indicator of cancer member of RAS oncogene family. It plays a key role in RAS/ MAPK signaling which involved in proliferation, differention and apoptosis Activating mutations of the K-RAS gene impairs GTPase activity. RAS mutations involves codon 12 0r 13 causing gly -> asp
COLD-PCR stands for co-amplification at lower denaturation temperature-PCR
=> enriches variant alleles from a mixture of wild-type and mutation-containing DNA
Principle : single nucleotide mismatch anywhere along a double stranded DNA sequence alters the Tm
(0.2-1.5%) of that sequence.
Advantages : Increase the sensitivity of detection of variants. Didn’t require additional costs in either reagents or instruments. Single-step method capable of enriching both known and unknown minority alleles irrespective of mutation type and position
Disadvantages : Optimal Tc must be measured and determined for each amplicon.
Downstream applications using conventional PCR that can be replaced by COLD-PCR:
COLD-PCR is used for applicable for the detection of the K-RAS mutations
COLD-PCR
qRT-PCR
Sanger sequencing
MALDI -TOF
RFLP
Principle of MALDI-TOF :
Microsatellite instability: Appearance of abnormally long or short microsatellite in DNA.
It includes – microsatellite alterations and loss of heterozygosity
Methylation of CNAs :
Cancer Methylated gene Indicator
Colorectal CDKN24 Prognosis, tumour grading & Dukes staging
Prostate RAR-beta -
P16 gene High storage disorder
Lung/ Breast RASSF1A -
Detected by MS-PCR
Ig gene Rearrangements
Circulating miRNA as a potential Biomarker :
Tumour Entity miRNA marker References
B-Cell Lymphoma miRNA-155,miRNA-210, miRNA-21
Lawrie et al.
Breast Cancer miRNA-195, miRNA-155 Heneghan et al.
Colon Cancer miRNA-29, miRNA-92a Huang et al.
Ng et al.
Gastric Cancer miRNA-17-5p, miRNA-21, miRNA-106a,miRNA-106b
Tsujiura et al
Leukemia miRNA-92a Tanaka et al.
Lung Cancer miRNA-25. miRNA-223 Chen et al.
Oral Cancer miR-31 Liu et al.
Ovarian Cancer miRNA-126, miRNA-93 Resnick et al.
Pancreatic Cancer miRNA-210 Ho et al. ,Wang et al.
Present disadvantages as Cancer Biomarker :
Presence of altered CNA is not informative of the site of the tumour.
Unclear that whether all cell release CNA at a same rate.
Chorionic villus sampling (CVS)
Amniocentesis
Invasive Non-invasive
Not possible before 11 week gestation.
CNAs in pregnancy
Circulating DNA in pregnancyIn 1997 , Lo et al. discovered circulating fetal DNA in the maternal blood
They found that fetal DNA concentration increases 3.4% & 6.2% of total DNA in maternal plasma in early and late gestation respectively.
fetal DNA
Maternal Plasma
Cause of fetal DNA in maternal plasma: Lysis of circulating fetal cells by maternal immune system. Apoptosis of cells during fetal development. Apoptosis of trophoblast.
Advantage of fetal DNA/RNA detection :Advantage of fetal DNA/RNA detection :
Detectable from 5Detectable from 5thth week gestation week gestation
Presence of fetal DNA/RNA allows the potential of using cell free maternal plasma/serum for Non-invasive prenatal diagnosis.
Diagnosis of chromosomal aneuploidies.
Fetal RhD status.
Sex-linked Disorders.
Circulating RNA as a Biomarker :
Circulating RNA and miRNA are detected in maternal plasma.
Placental-mRNA is specific and hence it’s presence is a analysis of pregnancy.
mRNA of hPL and hCG are detected in the plasma by qRT-PCR.
miRNA are also elevated
eg. miR-141 miR-149, miR-299-5p, miR-135b , miR-517A
Both DNA & RNA are cleared from circulation within 30 min. of delivery
Circulating fetal nucleic acid detection in the past decade :
Quantitative Quantitative measurementmeasurement
Qualitative Qualitative measurementmeasurement
Detection of small sequence difference by Mass spectroscopy
Detection of new classes fetal CNA marker eg. DNA methylation marker
Absolute quantification of fetal CNA by digital-PCR
Detection of fetal chromosomal aneuploidy by RNA-SNP analysis
10%
Placenta maternal plasma maternal blood
Limitations :-Low fetal DNA concentration. Interference by high maternal DNA background.
RNA-SNP Allelic ratio
DNA is diluted into multi-well plates
Amplification by PCR
Analysis of PCR products of mutants and
WT sequence by fluorescence probes
The data are recorded as positive or
negative represented as 0 or 1
Well with positive signal shows one
template molecule
Bisulfite Sequencing:
Treatment of DNA with bisulfite treatment converts Cytosine residues to Uracil but leaves 5-methyl cytosine residues are unaffected.
Sulphonation Hydrolytic deamination Desulphonation
Cytosine Cytosinsulphonate Uracilsulphonate uracil
Methylation-Specific PCR
Unmethylated Unmethylated Methylated Methylated Fetal maternal Fetal maternal Allele Allele Allele Allele
Fetal DNA Maternal DNA
Maternal plasma
A G A GBisulfite conversion
Bisulfite conversion
MSP-Primer of unmethylated sequence U-for U-rev
MSP-Primer of methylated sequence M-for M-rev
A/G genotyping by direct sequencing or primer extension
Stroke
Stroke involves a cascade :
Cerebral ischemia ->altered cerebral blood flow ->inflammation
-> production of ROS
Apoptosis
NecrosisRelease DNA/RNA
Rainer et al.
By Measuring the amount of CNAs it is possible to determine mortality and morbidity of patient.
it is a quick and non-invasive way.
q RT-PCR
Conclusions : Elevated Circulating nucleic acid are found in Cancer , Pregnancy, Trauma, Stroke. CNAs acts as a biomarker and allows Non-invasive methods of quantification. At present CNAs has disadvantage of lack of specificity. There is a scope of further research to nature and structure of plasma CNAs and related materials.
References :- Mandel, P. & Métais, P. Les acides nucléiques du plasma sanguin chez l‘homme. C.
R. Acad. Sci. Paris 142, 241–243 (1948). Nawroz, H., Koch, W., Anker, P., Stroun, M. & Sidransky, D. Microsatellite
alterations in serum DNA of head and neck cancer patients. Nature Med. 2, 1035–1037 (1996).
Swaminathan, R. & Butt, A. N. Circulating nucleic acids in plasma and serum: recent developments. Ann. N. Y Acad. Sci. 1075, 1–9 (2006).
Stroun, M. et al. The origin and mechanism of circulating DNA. Ann. N. Y Acad. Sci. 906, 161–168 (2000).
Gahan, P. B. & Swaminathan, R. Circulating nucleic acids in plasma and serum. Recent developments. Ann. N. Y Acad. Sci. 1137, 1–6 (2008).
Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C,Beljanski M. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 1989;46:318–22
Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumor DNA in blood Žplasmarserum. of cancer patients. Cancer Metastasis Rev 1999;18:65–73.
Chen Xq, Stroun M, Magnenat JL, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996;2:1033–5.
Ng EKO, Tsui NBY, Lam NYL, et al. Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals. Clin Chem 2002;48:1212–7.
Garcia-Olmo DC, Ruiz-Piqueras R, Garcia-Olmo D. Circulating nucleic acids in plasma and serum (CNAPS) and its relation to stem cells and cancer metastasis: state of the issue. Histol Histopathol 2004;19:575–83.
Lo YMD, Tein MSC, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.
Poon LLM, Leung TN, Lau TK, Chow KCK, Lo YMD. Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem. 2002; 48: 35-41.