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.

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