STR Profiling Matching Criteria: Establishment and Importance of a Cell Line Database

Transcription

1 STR Profiling Matching Criteria: Establishment and Importance of a Cell Line Database Margaret Kline Applied Group Biochemical Science Division Chemical Science and Technology Laboratory National Institute of Standards and Technology U.S. Department of Commerce Presented at: The ASCB 49 th Annual Meeting: December 6, 2009; San Diego CA As part of the Cell Line Workshop: Development of a consensus Standard for the Authentication of Human Cell Lines: Standardization of STR Profiling

2 Disclaimers Points of view are those of the presenter and do not necessarily represent the official position or policies of the US Department of Commerce. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.

3 Outline Components of an Ideal database Modeling the databases of the Forensic DNA Community Recommendations: Allele nomenclature Stutter vs allele call Heterozygous Peak height balance/imbalance Number of loci needed to ID different cell lines Issues seen: Reviewing data available on the web Reviewing historic electropherograms

4 Components of an Ideal International Cell Line Database All cell lines analyzed by the same STR loci. Instrumentation used for STR analysis capable of 1 base pair resolution. Use of a standard set of allele nomenclatures. Standard set of STR interpretation guidelines used for all profiles/allele calls. Peak height data included with the allele calls. Laboratory use of reference materials for method validation.

5 Progress Toward a Database The BioSamples database (NCBI) is under early stages of development/still under discussion phase. Cell line registration with genetic profiles is something it could contain. Stay tuned for more developments.

6 Forensic DNA Typing as a Model In the USA the Scientific Working Group on DNA Analysis Methods (SWGDAM) has established Short Tandem Repeat (STR) Interpretation Guidelines. These guidelines are laboratory based; that is, the laboratory is responsible for establishing and validating detailed criteria for assuring the STR profiles are reliable.

7 Factors that Forensic DNA Labs Consider Peak Amplitude analytical threshold Interpretation (stochastic) threshold Use of Controls reagent blanks, amplification blanks, positive controls Allele Designation comparison to allelic ladders designation of incomplete repeats (variant alleles x.1) alleles larger or smaller than allelic ladders Interpretation single profile presence of a mixture These are all quality assurance parameters

8 STR Core Loci for DNA Databases US Loci D3S1358 D8S1179 D16S539 D18S51 D21S11 FGA TH01 vwa CSF1PO TPOX D5S818 D7S820 D13S317 UK loci D3S1358 D8S1179 D16S539 D18S51 D21S11 FGA TH01 vwa Europeans are in the process of adding more loci. 8 loci in common plus amelogenin

9 So what s currently used by Cell Banks for STR typing? Cell banks are using a variety of STR typing methods including: PowerPlex 1.2: D16S539, D7S820, D13S317, D5S818, CSF1PO, TPOX, TH01, vwa and Amelogenin (XY). Second Generation Multiplex (SGM): D21S11, FGA, TH01, vwa, D8S1179 and D18S51 and Amelogenin (XY). Homebrew Methods some with overlap to PowerPlex 1.2 loci Coriell STR methods have no overlapping loci

10 Data was obtained from DSMZ (German repository including ATCC, JCRB, and Riken) tentleft_id= entries JCRB (Japanese repository including Riken) entries ATCC (US repository) Database/tabid/174/Default.aspx 678 entries All are using either PowerPlex 1.2 or a homebrew of the same loci.

11 Interpretation Issue 11 Allele should be called

12 Should this allele really be called? Inconsistent calling of the alleles present in the samples Lot A Lot B vwa TH01 AM TPOX vwa TH01 AM TPOX Should not be called, probably a stutter product Lot A and Lot B appear to be from the same sample

13 Lot A STR Profiling Interpretation Lot B 14 allele called 14 allele not called Cell Name Lot No. D5S818 D13S317 D7S820 D16S539 vwa TH01 Amel TPOX CSF1PO 293 A 8,9 12,14 11,12 9,13 16,19 7,9.3 X 11 11, B 8, ,12 9,13 16,19 7,9.3 X 11 11,12 Two different lots of the same cell line with different STR types at D13S317. Differences in STR types could be due to a change in the calling of the alleles as is illustrated by the electropherograms above.

14 So why can t we use the STR interpretation guidelines established by the Forensic DNA testing Community? Because a significant number of cell line DNA samples are just not the same as DNA obtained from a single human! Cancer cells are known to demonstrate a loss of heterozygosity at STR loci (loss of an allele). Hyperploid cell lines exist, potentially leading to within locus imbalanced peak heights!

15 Balanced peak height ratios Forensic labs are looking for balanced peak height ratios. That means with heterozygous loci we are look for the peak heights to normally be within 60 % to 100 %.

16 Imbalanced peak height ratios

17 Is There a Mixture present? Forensic labs look for: Peak imbalance (can not use that one alone). The presence of three or more alleles at one or more loci. Detection of a mixture can be dependent on: the amount of DNA amplified the number of loci examined

18 Tri-Allelic This cell line was derived from umbilical core vein. Both mother and child could be present explaining the allele imbalance and the tri-allelic patterns seen This is a mixture (as expected?)

19 Lot to Lot mutation - D7S820 Lot No. D5S818 D13S317 D7S820 D16S539 vwa TH01 Amel TPOX CSF1PO , ,10 9,12 17,18 7 X,Y 11 11, a 10, ,9.3,10 9,12 17,18 7 X,Y 11 11, b 10, ,9.3,10 9,12 17,18 7 X,Y 11 11, c 10, ,9.3,10 9,12 17,18 7 X,Y 11 11, a 2007b 2007c The sequence of this locus nominally has 9 T s found 14 base pairs down stream from the repeat. A loss of one T is known to occur to form the x.3 allele.

20 Sequenced D7S820 mutation allele Allele 6.3 has [GATA] 7 and a deletion of T 14 bp downstream of the repeat [GATA] 7 Del T [GATA] 12 Allele 12 Sequencing of this sample having the D7S820 was done as part of the variant allele sequencing project.

21 Variant Allele sequencing project For the past several years, our group at NIST has been sequencing unusual STR alleles found by members of the human identity testing community. Some of these alleles possess point mutations in primer binding sites leading to null alleles while others are "off-ladder" variants. Sequencing these unusual alleles helps to reveal the molecular basis for their variation. Variant reports are given back to the submitting agencies and with permission are posted on:

22 Incomplete 3 (+A) nucleotide addition Can be a result of too much DNA amplified in the PCR reaction (or too little polymerase and other PCR reagents). This type of PCR artifact should be avoided

23 Subline: has reportedly lost its Y Chromosome & one D5S818 allele Locus names D5S818 D13S317 D7S820 D16S539 VWA TH01 AM TPOX CSF1PO 11,12 10,11 10,12 12,12 16,18 9.3,9.3 X,Y 8,8 11,12 11,11 10,11 10,12 12,12 16,18 9.3,9.3 X,X 8,8 11,12 Submission Subline X Y While there is a more pronounced imbalance between alleles at these loci the alleles not listed in the subline genotype are present.

24 Different Names Same Type Matching is probably OK Cell name Locus names D5S818 D13S317 D7S820 D16S539 VWA TH01 AM TPOX CSF1PO COLO ,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 COLO201 10,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 COLO ,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 COLO205 10,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 COLO-206F 10,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 NS-3 10,13 10,12 9,10 12,13 15,15 8,9 X,X 11,11 11,12 COLO 205 The line was derived from tissue from the same patient as COLO 201 COLO 206F The cell line was established from the same patient as the cell lines COLO 201 and COLO 205 NS-3 Human gastric cancer cell line maintained in nude mice. Cross contamination with the COLO 201 found (12/15/2003)

25 Same Cell Line Name Different STR Profiles Cell Locus names # name D5S818 D13S317 D7S820 D16S539 VWA TH01 AM TPOX CSF1PO A TALL-1 9,12 8,9 10,11 9,10 17,19 7,9.3 X,Y 8,11 11,13 B TALL-1 9,12 8,9 10,11 9,10 17,19 7,9 X,X 8,11 11,13 C TALL-1 9,12 8,9 10,11 9,10 17,19 7,9 X,Y 8,11 11,13 Data of the TALL-1 cell line from 3 different Cell Banks Line A is Amelogenin XY but TH01 7,9.3 (possible typographical Error?) Line B is Amelogenin XX but online description is male origin, with TH01 7,9. Line C is Amelogenin XY but TH01 7,9. No electropherograms were available for review

26 Thoughts STR calling criteria need to be established and used consistently. Automated software calling (e.g., OSIRIS) Freeware! External review of allele calls from electropherograms Based on available data it appears that eight STR loci plus amelogenin are not enough loci to discriminate between closely related cell lines.

27 Thoughts A loss or gain of an allele detected by STR analysis may or may not be indicative of some other change occurring within the cell line. The possible change may or may not affect the functionality of the cell line At the minimum it indicates somewhat of a sub-clone / subline being established. Need to establish that the correct amount of input DNA was used to avoid stochastic amplification. Tracking the STR changes over time can be accomplished if the date of the establishment of the cell lines is tracked.

28 Thoughts There are cases where the name of the cell line is different from another line but the STR types are the same More STR loci would be needed to establish relatedness of these lines. Or if the cell lines were mislabeled at some point.

29 Contact Information Margaret Kline John Butler Group Leader first name. last nist.gov