CSF1PO: APPLICATIONS IN

FORENSIC SCIENCEALICAI MORALES, BECKY SMITH, KIMBERLEY MASIH, SUET FAR WONG,

TOBY VICKAR, AND YE JEE ROH

SHORT TANDEM REPEATS IN FORENSIC SCIENCE

• What makes a good STR marker?

• High heterozygosity among the population (>70%)

• Separate chromosomal locations

• Reproducibility of results when multiplexed

• Low stutter ratio

• Low mutation rate

• Allele sizes of STR marker should fall within the range of 90-500 base pairs

HISTORY OF CSF1P0

• CSF1PO is seen as having moderate to low discrimination power

• CSF1PO is commonly used today due to it being one of the 13 CODIS core loci

• It was one of the first STRs ever discovered

BACKGROUND OF CSF1P0 – LOCATION

• Located on chromosome 5

(5q33.3-4)

• Size range of allelic ladder varies

based on kit/primer used

CSF1P0

BACKGROUND OF CSF1PO

• Simple repeat STR: Contains units of identical length and sequence

• CSF1PO in humans (HUMCSF1PO) is a proto-oncogene for the CSF-1 receptor gene

• The common sequence motif in the 5’ 3’ direction is “AGAT”

• It is the number of repeats of "AGAT" at CSF1PO that varies from one person to another on each inherited chromosome

http://www.cstl.nist.gov/strbase/str_csf.htm

BACKGROUND OF CSF1P0: PROS AND CONS PROS

• Tetranucleotides are the most commonly used STRs in Forensic Science because of their low stutter ratio (compared to di- or tri- STRs)

• Low mutation rates

• High amount of population research done already for this STR

• Narrow allele size (which allows: multiplexing, reduces allelic dropout from preferential amplification of smaller alleles, usage of degraded DNA, reduces stutter product formation in mixture samples)

CONS

• CSF1P0 typically exhibits the least amount of variation between individuals

• Not used nowadays in Forensic Laboratories in Europe

STUTTER

• Stutter peak: minor allele peak with repeat units shorter or longer than parental allele peak

• Due to slipping of Taq polymerase during PCR

• Stutter ratio: height/area of stutter peak over that of allelic peak

• For lower stutter level:

Longer repeat units

Complex repeat unit sequence

Shorter allelic length

• Ratio should be less than 0.15 to be considered a stutter peak

STUTTER OF CSF1PO

• 319 single source DNA samples

• Mean stutter rate of CSF1PO was 5.83%• Lower than vWA and FGA, but higher THO1 and TPOX

AmpflSTR Profiler DNA Typing System

• Minimum of 100 data points from population database samples• Mean stutter percentage of CSF1PO fell within the 8% stutter

AmpFlSTR® Identifiler PCR™ Amplification Kit

• Stutter filter percentage calculated for CSF1PO is 8.77%• How % is calculated: Mean stutter for the locus plus three standard

deviations• Peaks in the stutter position below the stutter filter percentage> filtered off

GlobalFiler PCR Amplification Kit

GLOBALFILER PCR AMPLIFICATION KIT

Thermo Fisher Scientific Inc., 2015

STUTTER OF CSF1PO

• Stutter percentages from internal validation of different kits under threshold of 0.15

• Shorter length of repeat unit and simple sequence of CSF1PO contribute to higher stutter ratio compared to other STR markers

• Also influenced by extrinsic factors such as primer designs used and reaction conditions

• Smaller stutter ratio would be more advantageous in interpretation of sample profile

GlobalFilerTM PCR Amplification Kit User Guide (2015).

PATERNITY TESTING

• Has low mutation rate (0.16%): this is good for paternity testing

• Research has been done to improve the reliability of the use of CSF1PO in paternity testing

• A recent study used CSF1PO and five closely linked SNPs (SNPSTR) and looked at their potential uses in paternity testing

• Found that paternity probability increased to 99.998%

Balloch et al. (2008)

COMBINED DNA INDEX SYSTEM (CODIS)• Includes 13 STR loci, plus Amelogenin

• Why was CSF1PO selected as one of these 13?

• CSF1PO and D5S818 are the only two CODIS STRs that share a chromosome

• Recent studies show that CSF1PO does not have a very high heterozygosity rate

• Looking back in the literature to when CODIS was first constructed, CSF1PO was said to be one of the more discriminating STRs (Hammond et al, 1994)

• More current research shows that CSF1PO has the second lowest heterozygosity rate of all CODIS STRs (Planz et al., 2012)

• In most comparative studies of CODIS vs. Non-CODIS STR’s, better results were found when non-CODIS STR’s were used

http://www.cstl.nist.gov/strbase/fbicore.htm

POPULATION DATA

HETEROZYGOSITY RATES

Country Heterozygosity

Yugoslavia 0.748

South Korea 0.716

Mexico 0.7062

East Malaysia 0.714

Brazil 0.72304

Colombia/Antioquia 0.687

Serbia and Montenegro 0.68

China (Fujian province) 0.7208

Russia (Vulga-Ural) 0.743

Egypt 0.67

Italy 0.67

SUMMARY

• Overall, we would NOT add CSF1PO to our existing kit

• Due to a combination of low heterozygosity rates, low variation among alleles, and comparatively high stutter ratio, CSF1PO is among the least discriminating STR’s we have today

CSF1PO

REFERENCES• https://www.fbi.gov/about-us/lab/biometric-analysis/codis/codis-and-ndis-fact-sheet

• Hammond, H. A., Jin, L., Zhong, Y., Caskey, C. T., & Chakraborty, R. (1994). Evaluation of 13 short tandem repeat loci for use in personal identification applications. American Journal Of Human Genetics, 55(1), 175-189.

• Tsai, C. W. , Yang, C. H., Chou, S. L., Cheng, S. G., Pai, C. Y. (2013) Non-CODIS DNA markers could be more effective than CODIS-based STRs in problematic biological relationship cases.Romanian Society of Legal Medicine, (21), 245-248.

• Butler, J. (2005). Forensic DNA Typing: Biology, technology, and genetics of STR markers, Second Edition. Elsevier Academic Press. Pg. (104-108).

• D.Y. Wang et al. (2015). Developmental validation of the GlobalFiler Express PCR Amplification Kit: A 6-dye multiplex assay for the direct amplification of reference samples. Forensic Science International: Genetics , (19), 148–155.

• L.K. Boon et al. (2006). Internal validation of the AmpFlSTR Identifiler PCR amplification kit on the ABI PrismR3100 genetic analyzer for use in forensic casework at the Department of Chemistry, Malaysia. International Congress Series, (1288), 379–381.

• L. Blackmore, M.H. Luebke, J.M. Laird & P.J. Newall. (2000). Preferential Amplification and Stutter Observed in Population Database Samples using the AmpflSTR Profiler™ Multiplex System. Canadian Society of Forensic Science Journal, 33:1, 23-32.

• R. Li. (2015). Forensic Biology, 2nd Edition. USA: CRC Press.

• GlobalFilerTM PCR Amplification Kit User Guide (2015). Thermo Fisher Scientific Inc.

• K.J.D. Balloch, J. Marshall, J. Clugston, J.W. Gow, Reporting paternity testing results when 2 exclusions are encountered, Forensic Science International: Genetics Supplement Series, Volume 1, Issue 1, August 2008, Pages 492-493

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