Prof. W.

McLaughlin FSCI6603

Mixtures arise when h more than h one individual d d l contributes to the DNA stain Interpretation of forensic DNA mixtures is a challenging task in forensic casework Laboratories should conduct sufficient validation studies .

Common in cases of sexual assault where the source of DNA evidence can include:
th the victim, i ti the perpetrator(s), and partner(s) ( ) of the victim the consensual p

Even more complicated in incest cases

http://www.nfstc.org/pdi/Subject06/images/pdi_s06_m03_06.swf

It is also possible to find a mixture in a known blood sample from a deceased individual if that person was transfused prior individual, to death It is also possible to find a mixture when intimate samples are analyzed p y

Intimate samples are generally swabs collected from a persons body (skin, buccal, vaginal swabs)

possible allele overlap among an unknown number of contributors 3 ll l patterns 3-allele stochastic fluctuation with low quantity or degraded template the semi-quantitative activity of Taq polymerase

A stain exhibiting more than two alleles in a single DNA system shall be considered a mixed stain except in the case of genetic irregularities (e (e.g., g trisomy, trisomy somatic mosaicism, or duplication) If more than two alleles are observed in at least two DNA systems, the presence of a mixed stain shall be assumed

When there are three or more alleles are observed at multiple loci Notable differences in the allele intensities (peak heights) are detected in a STR profile

The presence of more than two alleles at any one locus A peak ki in a stutter position ii that h h has a higher hi h RFU than what would be expected, based upon the established stutter percentage for that locus (>50 RFU) Imbalanced and/or peak height / unexpected p p g percentages

No obvious major contributor with no evidence of stochastic effects

Class A C

Stochastic effects S

Clearly distinguishable major and minor DNA components; consistent peak height ratios of approximately 4:1 (major to minor component) across all heterozygous systems, and no evidence of stochastic effects

Mixtures with no major component(s) and evidence of stochastic effects

Class B C

Class C C

(FGA locus) l ) represent a clear major and minor contributor (vWA locus) represent an ambiguous bi minor i contributor

vWA major contributor masking ki minor i contributor t ib t

STR Typing Results of the Forensic Evidence (Sperm Stain) and the Reference Samples from the Victim and Suspect*. Locus Victim Semen Stain Suspect D3S1358 15, 19 15, 17, 19 15, 17 TH01 9 9 9, 9.3 3 9 9 9, 9.3 3 9 9 9, 9.3 3 D21S11 31, 32.2 29, 30, 31, 32.2 29, 30 D18S51 12, 16 12, 16, 18 12, 18 Penta E 11, 14 11, 14, 17 14, 17 D5S818 11 12 11, 11 12 11, 11 11 11, D13S317 11, 11 11, 13 11, 13 D7S820 11, 12 9, 11, 12 9, 11 D16S539 6S539 11, , 11 10, 0, 11 10, 0, 11 CSF1P0 12, 13 11, 12, 13 11, 13 Penta D 11, 12 11, 12 11, 12 VWA 17, 18 15, 17, 18 15, 17 D8S1179 10 10 10 10, 13 10, 13 10, 13 TPOX 8, 8 8, 8 8, 8 FGA 21, 23 21, 23 23, 23 g X, X X, Y X, Y Amelogenin *Alleles given in bold could be assigned to the suspect upon re-analysis

In general, the presence of not more than four alleles system allows the ll l in i a given i ll h assumption i of at least two independent stain donors In general, the presence of not more than six alleles in a given system allows the assumption of at least three independent stain donors In general general, if more than six alleles are observed in a given system, the exact number of stain donors cannot be reliably determined

No universal formula used to interpret and report mixtures Once the determination of a mixture has been made made, it may be helpful to use the case information and/or sample type to aid in the interpretation of the sources of the mixture p

It is common to obtain samples where one of the contributors (e.g., the victim) is known. In these cases, cases it may be possible to infer an unknown profile by subtracting the contribution of the known donor from the mixed profile
http://www.nfstc.org/pdi/Subject06/images/pdi_s06_m03_06_b.swf

Allele 19* 20 23 25*

Peak Height 250 674 717 225

Assuming only two donors, the mixture proportions are estimated by summing the peak heights of the two major or minor alleles and dividing by the peak heights of all four alleles. This is then multiplied by 100 to provide a p p percentage g contribution.
250 + 225 / 250 + 674 + 717 + 225 = .25 x 100 = 25% contribution from the minor donor 674 + 717 / 250 + 674 + 717 + 225 = .75 75 x 100 = 75% contribution from the major donor

This can also be represented as a ratio by summing the peak heights of the two minor alleles and dividing them by the sum of the peak heights from the two major alleles
250 + 225 / 674 + 717 = .34 or a ratio of approximately 1 to 3

A second check of this hypothesis is evaluation of the peak height percentages. In this case the peak height percentage for genotype 1 (19, (19 25) is 90% and genotype 2 (20, 23) is 94% These values are within the acceptable range of peak height percentage for balanced heterozygotes

If the genotypes of two persons are ab and bc, then they share b allele. The contributions are assumed to be additive Can expect the proportions of a:b:c = 2:3:1

Applying statistical is A l i i i l interpretation i i i simple i l when h dealing with one contributor (e.g. single source samples or mixtures where a major component can be defined or inferred). Statistical evaluation of multiple contributors is more complex Th i f ll calculations l l i i h k l d of f The b basis for all is the knowledge the allele frequencies in the relevant population

Perform no statistical calculations : The suspect is included or excluded from being a contributor to the mixture observed Performing match probability estimations after deducing the possible genotypes of the contributors Using (PE) g exclusion or inclusion probabilities p or (PI) Performing likelihood ratio calculations (LR)

DNA stains from crime scenes are usually characterized through multi-allelic (STR) loci, so there is a need to investigate which approach is the most efficient in determining the number of individuals involved in a mixture

If all alleles of a person in question are uniformly present in a mixed stain, the person shall be considered a possible contributor to the stain

If alleles of a person in question are not present in a mixed stain, the person shall not be considered as a possible contributor to the stain

Inclusion

Exclusion

PI represents the combined probability (relative population frequency) of all combinations of genotypes that cannot be excluded to have contributed to the DNA profile of a stain PI is equivalent to the match probability in the case of a stain originating from a single person

It is equivalent to the probability that a randomly selected person is a contributor to the stain [=random man not excluded (RMNE)].

PI is independent of assumptions about the number of possible contributors to a stain, the genotypes, genotypes and the ethnic origin of persons involved in a given case Based on the criteria given for inclusion

The probability of exclusion provides an estimate of the proportion of the population that has a genotype composed of at least one allele not observed in the mixed profile The probability of exclusion PE=1PI indicates the probability of excluding a randomly selected person as a contributor to a given stain

PI is calculated from the sum of all genotypes of possible stain contributors Assuming g that allele frequency q y data conform to HardyWeinberg equilibrium PI = a2 + b2 + c2 + 2ab + 2bc + 2ac Assuming a frequency of 0.1 for all alleles PI 0.32 0.09 or 9%

Expected that 9% of a group of randomly selected persons will not be excluded as stain contributors ~ 1 of 11 randomly selected person (RMNE) The PE is calculated from the difference PE=1PI = 1- 0.09 = 0.91 or 91%

Expected that 91% of a group of randomly selected persons will be excluded as stain contributors

The likelihood ratio is the ratio of two probabilities of the same event under different hypotheses Thus for events A and B, the probability of A given that B is true (H1), ) divided by the probability of event A given that B is false (H2) gives a likelihood ratio

Based on the assumption of two mutually excluding hypotheses Requires the description of a distinct scenario for a given stain case Both hypotheses explicitly describe alternative scenarios for the origin of a stain

Likelihood ratios are usually constructed with the numerator being the probability of the evidence (E) if the identified person is the source of the evidence, evidence and the denominator being probability of the evidence (E) if an unidentified p person is the source of the evidence LR = L (EIH1)

L (EIH2)

P(E/H1) is the probability of the evidence given a presumed individual is the contributor P(E/H2) is the probability of the evidence given the presumed individual is not the contributor of the evidence

Given a two-person mixed stain M and that all ll observed b d alleles ll l can b be explained l i db by th the genotype of the victim, Gv, and the genotype p , Gs, , the hypotheses yp can be of the suspect, formulated as follows:
Hypothesis Hp (view of the prosecution): The stain M originates from the victim V and the suspect S Hypothesis Hd (view of the defense): The stain originates from the victim V and from a unknown person U unrelated to the suspect

LR =

L (MIHp) = L (MIGv,Gs) L (MIHd)

L (MIGv, , Gu)

The resulting LR provides a numerical value, which indicates how many times more likely the observed DNA profile is under the assumption of the scenario described in Hp compared to the scenario described in Hd

First derive the numerator of the LR. The prosecution claims that the stain can be explained l i db by a combination bi i of f the h genotypes of the victim and the suspect, as there are no unaccounted alleles Hence, the numerator results as L(MIHp) = L(MIGv,Gs) = 1

The defense, however, claims that the suspect has not contributed to the stain. The genotype of the suspect is not relevant since the presence of allele c in the mixture must be explained by the contribution of an unknown person. As allele c may have been contributed either by a person homozygous for allele c or from a person heterozygous for c in combination with allele a or b,

the denominator is as follows: L(MIHd) = L(MIGv,Gu) =2ac + 2bc + c2

LR =

1 2ac+2ab+ + c2

Assuming the freq = 0.1 for all alleles LR = 1 0.02+0.02+0.01 = 1 0.05 = 20

it is 20 X more likely to observe the DNA profile if the mixed stain originated from the victim and the suspect than if it originated from the victim and an unknown person (or a random individual from the population)

LR < 1 the genetic evidence has more support from the denominator hypothesis LR = 1 the h genetic i evidence id h has equal l support from both numerator and denominator hypotheses LR > 1 the genetic evidence has more support from the numerator hypothesis pp yp

The likelihood ratio is a ratio of probabilities, and can take a value between zero and infinity The higher the ratio ratio, the more likely it is that the first hypothesis (Hp) is true

Establish mixture interpretation guidelines to minimize potential bias that could be influenced by any reference sample analysed Important to establish QA protocols to include mixture studies prior to performing case work analysis using y g a new technology gy

Based on the evaluation of the profile in its entirety Distinguish true alleles from non-allelic artifacts (stochastic effects)
Stutter peaks ( )p peaks Minus A(-A) Pull-up peaks Off ladder alleles P kh i ht i b l Peak height imbalance

The RFU can be used to establish peak height and/or peak area The RFU can therefore be used the determine interpretable signals Therefore important to establish threshold values for fluorescent signals

Peak Amplitude Threshold (PAT) Defines the minimum peak height in RFU that confidently fid l ascribes ib a true PCR amplicon li peak k and when confidence is too low to reliably assign a peak as an allele PAT the lowest peak height value that the lab uses PAT above the lowest limit of detection (LOD)

Match Interpretation Threshold (MIT) MIT establishes the minimum peak height in RFU that h all ll amplicon li peak k must di display l to confidently conclude that no genetic components of the sample failed to be detected after PCR due to low template conc, degraded sample or inhibitors

The PCR process is not 100% efficient Non-allelic peaks such as stutter, pull-ups can be introduced into the process For an unbiased assessment of the potential allelic data is to do the peak assignments for the evidentiary profile(s) before working on the reference sample(s)

Allelic vs non-allelic determinations for the evidentiary profile will therefore not be influenced by any conscious or unconscious bias predicated on the DNA profile of the reference sample(s)

Although lh h uncommon can be b observed b d at a loci in a profile and can be from a single source Most likely interpreted as a mixture? Things to consider peak height imbalance Must be considered on a case by case basis

Estimate the minimum number of contributors Based B d on the h l loci i that h exhibits hibi the h the h greatest number of allelic peaks E.g. 5 alleles are detected at 1 or more loci, the DNA typing results are consistent with having arisen from 3 or more individuals

The STR typing result for exhibit Q1 is a mixture of DNA from three or more individuals or; The STR typing results for exhibit Q1 indicate the presence of DNA from at least three individuals

Fingernail debris (recovered as scrapings) was taken from a man accused of sexually assaulting his biological daughter by digitally penetrating her vagina.

Fingernail scrapings

VWA THO1 16,18 7,9.3

F13 5,7

Suspect D Daughter ht

VWA THO1 F13 16,18 7,9.3 5,7 16 16 9 16,16 9.3,9.3 393 5 5,7 7

Ladd et al Interpretation of Complex Forensic DNA Mixtures CMJ 42(3):244-246,2001 Mi t 42(3) 244 246 2001 P. Gill et al DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures. Forensic Science International 160 (2006) 90 90101 101 Budowle et al Mixture interpretation: Defining for the the relevant features for guidelines g assessment of mixed DNA profiles in forensic case work. J Forensic Sci 54: 810 821, 2009