Germline Mutation Analysis of TP53 Gene

in Li Fraumeni Syndrome(LFS)

Institute: Tata Memorial Centre Advanced Centre for Treatment,

Research and Education in Cancer (ACTREC)

Guide: Dr. Rajiv Sarin

Principal Investigator-Sarin Lab

ADITI MAJUMDAR
B.TECH (BIOTECHNOLOGY) 4th YEAR
AMITY UNIVERSITY MUMBAI

OTHER NAMES FOR THIS GENE
 Antigen NY-CO-13
 Cellular Tumor antigen p53
 P53 Tumor suppressor
 P53_HUMAN
 Phosphoprotein p53
 Transformation-related protein 53
 TRP53
 Tumor protein p53 (Li-Fraumeni
syndrome)
 Tumor suppressor p53
TP53 GENE: INTRODUCTION
NORMAL FUNCTION HEALTH CONDITIONS RELATED
TO GENETIC CHANGES
 The TP53 gene provides instructions for
making a protein called tumor protein p53
(or p53). This protein acts as a tumor  Breast cancer
suppressor, which means that it regulates
 Bladder cancer
cell division by keeping cells from
growing and dividing (proliferating) too
 Cholangiocarcinoma
fast or in an uncontrolled way.  Head and neck squamous cell
carcinoma
 When the DNA in a cell becomes damaged  Li-Fraumeni syndrome
by agents such as toxic chemicals,  Lung cancer
radiation, or ultraviolet (UV) rays from  Melanoma
sunlight, this protein plays a critical role in  Ovarian cancer
determining whether the DNA will be
 Wilms tumor
repaired or the damaged cell will self-
destruct (undergo apoptosis). If the DNA
 Other cancers
can be repaired, p53 activates other genes
to fix the damage. If the DNA cannot be
repaired, this protein prevents the cell
from dividing and signals it to undergo
apoptosis. By stopping cells with mutated
or damaged DNA from dividing, p53 helps
prevent the development of tumors. 2
 TP53 GENE

CHROMOSOMAL LOCATION

 Cytogenetic Location: 17p13.1, which is the short (p) arm of chromosome 17 at position 13.1

 Molecular Location: base pairs 7,668,402 to 7,687,550 on chromosome 17 (Homo sapiens Annotation
Release 109, GRCh38.p12) (NCBI)

Credit: Genome Decoration Page/NCBI

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 p53 Domain Structure

Adopted and modified according to Bernard et al.

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 p53 FUNCTION and
ACTIVATION

DP
DP

http://p53.iarc.fr/ 5
 Tumors associated with
p53 germline mutation

http://p53.iarc.fr/
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 LI FRAUMENI SYNDROME

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https://www.cancer.net/cancer-types/li-fraumeni-syndrome
 LI FRAUMENI SYNDROME
DIAGNOSTIC CRITERIA:

https://www.semanticscholar.org/paper/TP53-and-CDKN1A-mutation-analysis-in-families-with-Andrade-
Santos/https://www.semanticscholar.org/paper/TP53-and-CDKN1A-mutation-analysis-in-families-with-Andrade-Santos/ 8
 TP53 Gene
OBJECTIVE: Germline Mutation Analysis of TP53 Gene in Li Fraumeni Syndrome

- DNA Extraction using commercially

- Mutation Analysis available column

- Analysis of the purity and yield of DNA

- Clean-up by using Exo-SAP MATERIALS
using Nano Drop technique
- Sanger Sequencing AND
METHODS
- Polymerase Chain Reaction
1. Initial Denaturation – 95°C for 5 minutes;
2. Denaturation – 95°C for 45 seconds
3. Primer annealing – For exon 3 to 9: 63.3°C - Agarose Gel Electrophoresis
for 45 seconds
4. Extension – 72°C for 45 seconds; Repeat
step 2 – 4, 34 times
5. Final Extension – 72°C for 5 minutes
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 MATERIALS AND METHODS
Patients

 All patients were participants of studies approved by the Tata Memorial Centre-ACTREC Institutional Review Board.

 Written informed consent was obtained from all subjects for biobanking and genetic analysis. For minors, the written
informed consent was provided by the parents.

 All experiments were carried out in accordance with the approved guidelines and regulations.

 Germline mutations were examined in cancer patients with personal or family history suggestive of hereditary LFS or
LFL syndrome. These LFS/LFL families were enrolled in the Cancer Genetics Clinic of the Tata Memorial Hospital for
genetic counseling and genetic testing.

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Genetic Analysis
 WORKFLOW

 Genomic DNA was extracted from peripheral blood lymphocytes by Qiagen column according to the manufacturers protocol.
 The DNA binding domain (DBD) region of TP53 gene was sequenced by Sanger Sequencing.

Polymerase Chain Reaction

 For PCR amplification before Sanger Sequencing, we started with the commonly used primer set i.e., 3 to 9.
 Annealing temperature for exons 3+4, 5+6, 7, 8+9 were 63.3°C, for the PCR amplification.
 PCR were set up in 25 ul volume with 10X PCR buffer (2.5 ul), 2.5 mM dNTPs (1 ul), 10 pmol primers (0.5 ul each), 20 ng/ul
of gDNA (5 ul) and 2 unit of Taq polymerase (0.5 ul).

Sanger Sequencing

 Amplified products were cleaned with Exonuclease and Shrimp Alkaline phosphatase for sequencing.
 2 ul of cleaned PCR products with 1 pmol of primer is taken for cycle sequencing reaction.
 Post cycle sequencing products were sequenced with Big Dye Terminator kit on DNA sequencers 3500 Genetic Analyzer 8
capillary or 3730 DNA Analyzer 48 capillary.
 Chromatograms generated were analysed using Chromas Lite Version. 2.01 and Finch TV Version 1.4.0 software and
matched with reference sequences to identify the germline mutations.
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 RESULT

 18 probands were studied for the presence of mutation in the TP53 gene that is present on chromosome 3 to 9.
 DNA extraction using lymphocytes were electrophoresed using agarose gel electrophoresis

gDNA extracted from blood loaded on 0.8 % agarose gel, showing clear bands under UV transilluminator

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 POLYMERASE CHAIN REACTION

1. PCR Standardisation: The result for the PCR amplification is as follows:

EXONS PCR CONDITIONS RESULT
3+4 PCR Amplification 631 bp band amplified
5+6 carried out at 550 bp band amplified

7 63.3°C 283 bp band amplified

8+9 455 bp band amplified

2. PCR:
I. DNA extracted (from samples) was amplified using PCR from 18 patients suspected of having Li Fraumeni syndrome. For 3
to 9 exons involved in Li Fraumeni syndrome, PCR amplification was performed. The amplified PCR products were then
loaded and electrophoresed on 1% agarose gel. The gel was observed under the Gel documentation system after the run was
complete. It was observed that fluorescent bands of different intensities depended on the PCR product amplification. For
exons that had not previously shown a band, the PCR amplification was repeated.
II. The amplified PCR products were further diluted to a required concentration and were used for mutation analysis using
Sanger sequencing.
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 SANGER SEQUENCING

Single Nucleotide Polymorphism (SNP) c.215C>G, p.P72R in exon 3+4 of TP53 gene

Normal Sequence for this

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Germline mutation c.766A>C, p.T256P in exon 7 of TP53 gene

Normal Sequence for this

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Sample No. 15; c.766A>C, p.T256P

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Germline mutation c.818G>A, p.R273H in exon 8+9 of TP53 gene

Normal Sequence for this

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Sample No. 5 c.818G>A, p.R273H

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 CONCLUSION

• 18 probands suffering from Li Fraumeni Syndrome were tested on the basis of genetic testing.

• DNA was successfully extracted from the lymphocytes present in the blood sample.

• The quality and quantity of the extracted DNA was determined by electrophoresing the samples on
0.8% agarose gel.

• PCR standardization for exons 3 to 9 of the TP53 gene was carried out using normal DNA.

• The extracted DNA samples were amplified using PCR and were electrophoresed using 1% agarose
gel.

• The amplified PCR product having sufficient concentrations were subjected to Sanger sequencing.

• 2 cases were detected to harbour a missense mutation at c.766A>C, p.T256P in exon 7 and
c.818G>A, p.R273H in exon 8+9 of Tp53 gene. And 1 case was detected to harbour a single
nucleotide polymorphism at c.215C>G p.P72R in exon 3+4.

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 REFERENCES

[1] Bates S, Phillips AC, Clark PA, Stott F, Peters G, Ludwig RL, Vousden KH. (1998) p14ARF links the tumour
suppressors RB and p53. Nature 395:124-125

[2] Bell S, Klein C, Muller L, Hansen S, Buchner J. (2002). p53 contains large unstructured regions in its native state. J
Mol Biol, 322:917-927

[3] Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A,
McCormick F. (1996). An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science,
274:373-376

[4] Blagosklonny, MV. (2002). P53: An ubiquitous target of anticancer drugs. International Journal of Cancer, 98:161166

[5] McCormick F. (2001). Cancer gene therapy: fringe or cutting edge? Nat Rev Cancer, 1:130-141

[6] Strachan T, Read AP. (1999). Human Molecular Genetics 2. Ch. 18, Cancer Genetics

[7] Vogelstein B, Lane D, Levine AJ. (2000). Surfing the p53 network. Nature, 408:307-310

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Thank You