GOOD MORNING

VALIDATION OF SOMATIC MUTATIONS IN CERVICAL CANCER

REPORTED IN NGS SEQUENCING BY SANGER SEQUENCING

By N.N.P.KUMAR (KVPY FELLOW)

Under the Guidance of Dr. Rita Mulherkar,

Principal Investigator,Head, Dept. of Genetic Engg

Place of work :- Advanced center for treatment, Research & Education in

Cancer (TMC)

Contents :-• Introduction• Cervical cancer and carcinogenesis• NGS Sequencing• Validation by Sanger sequencing• Flow of work done• Results• Analysis• Bibliography

Introduction :-

• Cancer of the cervix is the third most common cancer among women worldwide after breast and colorectal cancer (Singh 2012).

• Cancer of the cervix is the most common female about 500,000 women are effected and About 300,000 women also die from the disease annually (WHO/ICO 2010)

Cervical carcinogenesis

Genetic Alterations• Major etiologic factor for cervical cancer

carcinogenesis is HPV, the majority of women infected with HPV do not develop cancer. Also, the disease generally appears decades after initial exposure to HPV (Ma, Wei et al. 2000).

• The genetic factors are also a major cause of the familial aggregation in cervical cancer, but Still not strongly understand (Dan Chen et al. 2013)

NGS Sequencing (2011-2012 Illumina ) :-• The behind the NGS is similar to CE • NGS extends the process across millions of

reactions in a massively parallel fashion, rather than being limited to a single or a few DNA fragments

• They are capable of producing hundreds of gigabases of data in a single run

• Types of NGS Sequencing• Whole genome sequencing• Exome sequencing• Trancriptome sequencing

NGS platforms available are :• ROCHE/454 LIFE SCIENCES• ILLUMINA/SOLEXA• APPLIED BIOSYSTEMS/SOLiD

Draw backs of NGS

• next-generation sequencing analysis depend largely on the allelic fraction as well as coverage of the mutated residue in the tumor and normal sample. Therefore, the NGS-based somatic mutation detection is prone to erroneous calls (Meyerson, Gabriel et al. 2010; Robison 2010).

• The validation rate in these technologies has been reported to be more than 85% (Robison 2010) .

• Due to this reasons some other techniques are needed for validation

Validation process work flow

• Selection of Mutation

• Primer designing

• PCR reaction

• Agarose gel electrophoresis

• DNA Purification

• Sequencing parameters adjusting

• Sanger Sequencing

• Results Validation

• Conclusion

Primer Designing• specific primers were designed using

NCBI-Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

• The primer sequences flanked 200 bases upstream and downstream to the variation position resulting in amplicon size between 250-400 bp.

• The obtained primers in lyophilized state were reconstituted by adding 10mM Tris (pH 7.5).

S.no Gene Variation

Importance 

1 ABCG5 aCg/aTg Involved in the regulation of dietary cholesterol absorption(Mi-Hye Lee et.,al.2001)

2 BTNL8 gTg/gGg  Involved in immune regulation (Lucie Abeler-Dorner et.,al.2012)

3 GRAMD1A cCc/cGc  _

4 TTN Gtc/Atc Key component in the assembly and functioning of vertebrate striated muscles.(Gene cards data base) 

S.no Gene Variation Importance 

1 ABCG5 aCg/aTg Involved in the regulation of dietary cholesterol absorption(Mi-Hye Lee et.,al.2001)

2 BTNL8 gTg/gGg  Involved in immune regulation (Lucie Abeler-Dorner et.,al.2012)

3 GRAMD1A cCc/cGc  _

4 TTN Gtc/Atc Key component in the assembly and functioning of vertebrate striated muscles.(Gene cards data base) 

DNA ISOLATION

Genomic DNA is isolated from mammalian cells using a lysis buffer containing SDS, EDTA and Proteinase K.

The cell and nuclear membrane lysis is done by SDS and Proteinase K; EDTA helps in inhibiting DNases by chelating Mg2+ ions.

AMPLIFICATION OF GENOMIC DNA USING PCR

• The reaction was performed in DNA Engine® Peltier Thermal Cycler (BIO RAD). The PCR conditions used for amplification are mentioned in the below table

PCR Reaction Parameters

Agarose Gel Electrophoresis

• Agarose gel electrophoresis is performed to test the PCR Efficiency

PCR PURIFICATION

• After confirming amplification, the PCR product was purified using QIAquick® PCR Purification kit (Hilden, Germany) as per the manufacturer’s protocol.

• Nano drop readings are taken performed to test the Sequencing parameters

S.no sample Concentration(ng/l) 260/280 Ratio 260/230 Ratio

1 ABCG5(Tumor) 27.2 1.77 1.55

2 ABCG5(Blood) 22.0 1.82 1.75

3 BTNL8 (Tumor) 14.6 2.03 1.99

4 BTNL8 (Blood) 12.8 2.04 2.00

5 GRAMD1A2(Tumor) 19.2 1.91 0.88

6 GRAMD1A2 (Blood) 12.4 1.93 1.27

7 TTN (Tumor) 17.1 1.89 1.68

8 TTN (Blood) 15 2.07 1.21

Sanger Sequencing• The Sanger method or dideoxy/chain

termination method of DNA sequencing is gold standard for DNA sequencing

• It is widely used for the detection of single nucleotide variations.

• It relies on enzymatic synthesis of DNA in vitro in the presence of chain terminating inhibitors (the dideoxy nucleotides or ddNTPs).

• Automation of this technique involves labeling of each of the ddNTPs with a different color fluorescent dye.

• These dyes fluoresce at different wavelengths when excited by a laser.

• The resulting fluorescence is captured by a CCD camera in the machine and translated into an electropherogram, which is a diagram of colored peaks that correspond to the nucleotide in that location in the sequence

Results of Sanger Sequencing

Results and Conclusions

• All the validated somatic variations were checked for their presence in C33A, Caski, HeLa and SiHa cell lines by automated Sanger sequencing.

• 5 patient samples are taken for study

• It is the first one of these type of validation

Conclusion

• 16 (out of the 27 variations selected) were validated by Sanger sequencing (14 somatic and 2 germline).

• The validation frequency was about 60%.

• Identical mutations (as reported in COSMIC database) in DIDO1, RNF111 and EP300 gene were validated to be somatic in cervical cancer.

• 6 of the validated somatic variations were present in important candidate genes of MAPKinase pathway (FGF7, SOS2, TGFBR2, MAP3K3, FGF22 and RPS6KA6). Therefore, this pathway was identified among the highly mutated ones in case of cervical cancer.

• TTN is involved in Immune regulation

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