FZ4202 Assignment I - Forensic



FZ4202 Assignment I

Y chromosome is a unique portion of the genome that is passed only in the male lineage. Recent developments in the technology of DNA analysis have led to the greater use of Y chromosome polymorphisms. Write an analytical review of the current technological advances and uses of Y chromosome polymorphisms as a forensic tool. Outline the anticipated future technological developments in this field.

Table of contents

Title Page 1

Contents page 2

Introduction 3

Basic Science 3

Why Y-polymorphisms? 4

Y-STR’s 4

Y-SNP’s 10

Databases 13

Uses of Y polymorphisms 14

Future technologies and improvements 14

Conclusions 15

References 16

Introduction

More sensitive human DNA identifying kits, higher powers of discrimination, lower costs, easier use, higher throughput, less chance of contamination, more applications, more accurate, just a few of the demands that forensic scientists have been asking for since forensic DNA profiling entered the scene. And they got it. The initial VNTR’s who started the whole process were quickly replaced by STR’s around 1994 and now, ten years later, Y-STR multiplexes and megaplexes are being validated for use in court. Highly polymorphic SNP`s are being discovered around the clock which have a high potential of supporting or even replacing the STR’s. But before it will come that far, a lot more research will have to be carried out.

This assignment was extremely difficult due to the rate of speed that articles are being published. Nevertheless I tried to keep it up to date as much as possible and described the most recent projects I could find.

The essay is divided in chapters with the main ones describing Y-STR’s and Y-SNP’s. It starts off with a basic introduction of how Y polymorphisms are being formed and what the consequences are. The future developments of Y-DNA profiling are outlined in the final chapter.

Basic science

During the male meiosis phase of the cell division the exchange of base pairs between the X and Y chromosome is limited to the pseudoautosomal regions (PAR) and recombination takes place at the most distal short arm (PARI) and at the top of the Y-chromosome long arm (PARII) For most of its length (the non-recombining portion of the Y-chromosome) the Y-chromosome is male-specific and effectively haploid and is transmitted from father to son unchanged unless a mutational event takes place1. This has several advantages and disadvantages for our forensic purposes and it will be explained in the chapters below.

Why Y polymorphisms?

- Most criminals (over 90%) are male

- Mixtures can be analysed without differential extraction which separates the sperm cells containing male DNA from the female cells.

- Advantages in cases of rape committed by azoospermic and oligospermic individuals (1-2%, but will become more frequent because of vasectomy). These samples don’t contain any (or very few) sperm cells from which DNA can be analysed, but could contain epithelial cells from the male reproductive tract which can be picked up by Y-STR’s in contrast with autosomal male and female DNA.

- To obtain a profile after degradation of sperm cells in a vagina, even ≥ 48 hours post coitus

- Migration cases

- Anthropological kinship studies: The Y-STR haplotype differentiation is due to more recent events than the Y-SNP mutations. The observed patterns of Y-STR similarity may plausibly be related to particular historical events e.g. the Spanish occupation of Holland in the 17th century, the Viking invasion in several countries2…

- Add to Y-SNP phylogenetic studies

- Complement mtDNA studies

- Y-STR’s: paternity cases when the father is deceased.

Y-STR’s

An awful lot of research is presently being done on finding new STR and SNP markers. On the IV International Forensic Y-User Workshop in Berlin2 several new multiplexes were introduced. Up to now STR markers are more used for the identification of haplogroups in different populations then for the identification of individuals. Several companies (ABI, ReliaGene, Biotype, Serac, GKT and Promega) have produced there own multiplexes. These are validated but some of them are not on the market yet.

Where do these new STR’s come from?

Manfred Kayser recently published a paper3 on his new 139 new loci and presented the findings at the conference. He selected 475 Y-STR candidates with the Tandem Repeats Finder software which had 8 or more simple repeats. Loci with X-chromosomal homology were excluded which left him with 281. After a male and female PCR testing evaluation he still had 166 and after variability testing across haplogroups the final result was 139 new hyper polymorphic loci. The 166 loci could be divided in 78 simple and 88 complex loci. 166 plus the 53 previously known loci gives 219 known Y-STR’s and statistical research showed that when the number of repeats increased, the higher the variance was.

(GATA)8 < (GATA)8(GACA)4 < (GATA)12

John Butler selected 27 markers of these 139 and did some of his own research on it. He found that DYS 490, 504, 525 and 557 were duplicated somewhere on the Y chromosome and on the X chromosome and were excluded from the multiplex. DYS 632 and 575 were not highly polymorphic and were left out as well. 22 Markers still remained and these are now being put in a multiplex4.

Minimal and extended haplotype

Pascali et al.5 defined a minimal haplotype for court use which was endorsed by the ISFG (International Society for Forensic Genetics) for any laboratory participating in the YHRDatabase project.

The European extended haplotype is the same as the minimal haplotype + YCAII.

The US minimal haplotype was the same as the European extended haplotype but quite recently SWGDAM suggested changing the error prone YCAII marker to DYS438-4396.

Current Research Projects

In spite of the defined minimal and extended haplotypes more research was undertaken to improve the multiplexes in several ways. To analyse the original markers of the minimal haplotype it needed 2-3 multiplexes because of the different conditions for the PCR primers. Marker DYS385 has to be run separately sometimes in the PCR and YCAII (still in use in 2001) had to be run separately too in order to get the extended haplotype. The primers for these markers were available in the literature but had been designed originally for single plex systems and were not suited for multiplex systems. Therefore new primers had to be designed with stringent conditions in order to get a minimum of complications and a maximum of product result7. Every research team had its own ways of doing this but the requirements were all the same:

- The complex had to be able to be amplified in a single tube

- Incorporation of additional polymorphic markers to increase the power of discrimination

- No female amplification products

- Spacing between loci in same colour to allow additional undiscovered alleles to be accommodated

- Similar concentration of primers to produce balanced amplification products

sensitivity to less than 100 pg DNA

- Should be tested by simulated casework, able to discriminate between unrelated males and the frequency of occurrence should be as small as possible: population study is needed.

How was this done7?

1. Primers needed to have similar annealing characteristics in order to generate a balanced yield from all of the PCR products during simultaneous amplification.

2. Avoid X-chromosome homology that would yield signal in female DNA samples or impact PCR amplification

3. Screening for intramolecular primer-dimer and hairpin formation.

4. Primer purity was improved by reverse-phase HPLC (no more dye-blobs).

5. Avoid polymorphic nucleotides in primer binding sites through sequence alignments of multiple GenBank entries.

6. PCR product size ranges had to be optimized in each dye colour (prevent overlapping). This complicates data and leaves large gaps which are a waste of space. This can be done by moving primer positions in the flanking regions surrounding the Y-STR repeat segment.

Several multi- and megaplexes have been designed the past four years. From these I picked out two multiplexes that have been published plus one which has been published and validated by SWGDAM and two commercial kits.

A list of recent projects

- NIST 20 Plex Y-STR Rick Schoske & John Butler

- 21 Y-STR MegaPlex by Hanson 2004

- SWGDAM 19 plex Validation

- Butler et al. new 18 and 22 STR multiplexes

Commercialised kits

- 17 STR’s AmpFlSTR Y-Filer PCR Amplification Kit by ABI

- PowerPlex Y System by Promega

- ReliaGene Y-Plex ™ with 12 markers

- BioType, Serac and GKT

A summary of each project and identification kit is given below.

NIST 20 Plex Y-STR Rick Schoske & John Butler8

This multiplex was developed two years ago by Rich Schoske & John Butler8 by packing as many loci into each dye colour as possible. Leaving some gaps in between the known alleles made sure that undiscovered alleles amplified with the same dye would not overlap the known alleles after the multiplex was developed. After the minimum and maximum allele sizes were determined for each marker, a spread of 10 bp was left between the known alleles labelled with the same dye.

The allele calling is not done through conventional allelic ladders but is based on sizing bin windows of up to ± 1.50 bp8. This technique relies on direct measurement and uses Loens-Specific Brackets (LSB) which serve as an internal standard: one smaller and one larger in size than the common set of STR alleles. The flanking region of the LSB is identical to the STR alleles giving them the same electrophoretic properties and all three alleles pass the detector in order of size and a programme calculates the exact size of the allele in the middle and can designate a name to it.

In order to determine the match probability the ‘Counting Method’ was used: simply how many times a particular haplotype has been observed in a database of size N, limiting the minimal frequency estimate to 1/N. In addition, some of the Y-STR loci are not very discriminating, which means that it would be premature to use Y-STR profiles to populate large databases of convicted offenders due to the large number of fortuitous matches that would be obtained8.

Butler et al. 8gave several presentations on this topic and announced that validations studies would be performed, and eventually that it could be commercialised into a kit. This process has finished now and ABI has come out with the Y-Filer, Promega with the PowerPlex Y System. The kit was distributed to several laboratories for validation and will be on the market soon.

Last year they developed a new 11 Plex kit and now they are in the process of validating a multiplex of 22 and 18 STR’s combining the extended US haplotype and the 11 Plex they developed earlier. They also developed a SRM Standard Reference Material set containing 5 males and 1 female samples which were typed with the 20 STR Y-Plex for other laboratories to use for verification purposes that their assays were run properly with any primer set.

21 Y-STR MegaPlex by Hanson 20049

Also called Multiplex IV after MPI/II/III. Twenty one markers were included into the multiplex but 25 sites exhibiting allelic variation are amplified. One locus is bi-local and one is tetra-local which means that everybody can have 4 of 9 common alleles.

Markers included in the megaplex:

DYS 443-9 463-4 522 557 452-56 468 527 588 458 484 531

The power of discrimination was described as being 1/54.000.

SWGDAM 19 plex Validation

MPI & MPII developed by Ashley Hall & Jack Ballantyne 10 was validated by SWGDAM. The development of an 18 locus Y-STR system for forensic casework11 was the initial purpose of their research.

The following markers were included:

MPI: DYS 391/2/3 389I/II Y-GATA A 7.2 438 385

MPII: DYS19 425 388 390 439 434 437 Y-GATA C4

Y-GATA H4

Promega’s PowerPlex Y System with 12 Y-STR’s

The PowerPlex Y System1 incorporates the 9 loci from the European minimal haplotype plus the twp loci added by SWGDAM (DYS 438 – DYS439).

Promega has an own Y-chromosome haplogroups database on their website which allows users of the kits to determine the match probability.

ReliaGene Y-Plex ™

ReliaGene commercialised the Y-Plex ™ with 12 markers12. This multiplex has been taken of the market because of patent problems with Promega.

AmpFlSTR Y-Filer PCR Amplification Kit by ABI

This kit amplifies 17 loci in a single PCR reaction using 5-dye chemistry13. This kit can be used in combination with the new Quantifiler Y kit to provide an assessment of a mixture sample. It includes the European minimal haplotype plus 6 additional loci.

The discriminatory capacities of the kit: 98.3, 98.8 and 99.1 % for the Hispanic, Caucasian and African American populations respectively. It has a sensitivity of up to 125 pg of input DNA and can detect mixtures of male and female DNA greater than 1:1000.

BioType, Serac and GKT

Biotype14 developed a Mentype Argus Y-MH, containing all the markers from the European minimal haplotype. In addition to this Biotype developed a multiplex called Mentype Argus X-UL which amplifies 4 X-STR markers and amelogenin and was specifically designed for paternity testing and mother-son kinship testing. The match probability can be calculated like autosomal markers as the markers come from different linkage groups.

Serac, a company in Germany has produced two Y-STR kits called genRes DYSplex-1 and 2. These include 6 (+ amelogenin) and 5 markers respectively.

GKT from South Korea has 4 kits available: GeneKin Y-STR System I to IV ranging from 3 to 4 loci15.

Y-SNP`s

Until 1997 only 11 Y-biallelic markers were known. Over 200 Y-SNP`s were discovered by Peter Underhill (Stanford University) using dHPLC16.

Over 250 SNP’s have now been identified on the Y-chromosome7. They are most commonly used in the population genetics to try and identify populations according to their SNP profile. SNP’s have an advantage over STR’s being that the PCR amplicon needed to detect the SNP’s is much smaller in length. This is better for the typing of degraded DA as the backbone of the DNA strain can be damaged by several factors.

For the time being SNP analysis is not regularly used for the identification of individuals. It is being explored in phylogenetics research to determine the population history of several minority groups but the assays are not discriminating enough to identify each individual on its own without getting several hits in its own population.

SnaPshot is still the most favoured technique for the detection of SNP’s as it is a robust, reliable and sensitive technique. Several other techniques however are also used and some of them are explained below.

29 SNP Multiplex Kit

This kit was designed by Maria Brion et al. and presented at the workshop2.

A selection was done of candidate SNP’s and a single multiplex assay was set up. Conditions of the markers selected were the quality of the flanking primers, the predicted interactions between primers, X-chromosome homology and the primer sensitivity. Validation studies were performed to check for the kit’s accuracy and sensitivity. The amplicons ranged from 79 to 186 bp.

This assay can be used to put individuals into major population categories but to provide discrimination between closer populations, a new selection of markers will be required.

Signet™ Y-SNP Identification System (Marligen)

This system uses Luminex xMAP™ Technology17:

Human male DNA is amplified with PCR and labelled with a fluorescent tag. Hybridized to an array of allele-specific oligonucleotides immobilized on latex beads. Each bead is identified by a unique spectral address (colour). After hybridization the bead mixes are analyzed by the Luminex 100™ System that records the spectral address of each bead and the amount of fluorescent tag bound to it.

Over 40 SNP`s per sample can be done in one day. The SNP’s are amplified in 5 multiplexes which reduces costs and no washing steps are required to detect the 42 loci and the amelogenin marker.

The first multiplex Marker Mix 1 is to determine the root population while mixes 2 to 5 are region specific.

This kit was used by John Butler at NIST to type his reference samples with18.

Juan J. Sanchez (2003)

Sanchez et al. 19developed a multiplex to detect 35 Y-SNP`s with a lower limit of 100 pg DNA. They used publicised SNP`s but redesigned the primers. 25 primer pairs amplify 25 templates with 35 SNP`s analysed

P. Vallone & Butler 2004 17

The goal of their study was to determine the most informative Y-SNP`s from 50 SNP’s which had been described in the literature.

Vallone and Butler used 2 methods for the detection of these SNP’s:

- Primer extension with fluorescence detection (ASPE)

- Allele-specific hybridization using flow cytometry (= Signet Luminex): 5 diff. multiplex reactions with fixed markers (explained above). (ASH)

3 multiplexes of each 6 SNP’s were used for the ASPE method and 35 SNP`s were amplified with the ASH method.

Only 2 of the SNP’s were needed to cover 60% of the sample in the population data sets. Other markers were of no value to define it further on the population tree.

Inagaki 2004

Inagaki’s multiplex20 contains 15 loci distributed on blood type genes. The advantage of using these markers is that the allele frequencies have been well investigated over time. The sex marker in this multiplex uses a SNP difference between the amelogenin locus on the X-chromosome and an amelogenin-like locus on the Y-chromosome, used to determine sex. 37 DNA fragments are being detected together with 38 SNP`s

The STR and SNP amplifications occur in different PCR reactions but there is only one analysis necessary because of a newly designed multi-injection method.

Databases

4 Major databases are available to forensic laboratories:

1.

2.

3.

4.

Ybase and YHRD are the most valuable databases from the four listed above. Ybase is searchable on 49 markers while YHRD allows doing a neighbour search, meaning that one can submit a haplotype – 1 STR marker to look for common haplotypes with 1 STR marker less in common.

These databases are needed to determine the match probability in identification cases and to determine to which haplogroup a certain population or individual belongs.

Currently the YHRD database consists of over 25,000 haplotypes in a worldwide set of 229 populations. The continuation of submitting haplotypes from all over the world will permit more accurate grouping and mapping of haplotypes. (EMPOP which is a database for mtDNA haplotypes will be launched soon22.)

Problems with these databases are of course what population one should take to estimate the match probability. E.g. To determine the population group of a person from the UK, are we going to use the European database, a more local one or are we going to search via his surname?

Uses of Y-polymorphisms

STR’s and SNP’s can be used to identify individuals. Especially in mixed stains as outlined above these markers can be very helpful to determine the male component.

So far the SNP’s have only been used to determine haplotypes within haplogroups with the aid of the STR markers but as more and more robust and discriminating multiplexes will become available these too might be used for the identification of individuals.

Studies which have been carried out with Y-SNP`s so far only examine similarities or differences between populations rather than to evaluate the ability of SNP markers to distinguish between individuals in the same population.

The scientific committee needs to do more studies; otherwise it may be difficult to assess which markers will be useful in forensically relevant datasets if these markers are going to be used for human identity applications.

Butler states that if Y-SNP`s are used at all in future forensic applications, it will likely be in conjunction with Y-STR information.

Sanchez19 on the other hand is convinced of the use of SNP’s and says that they will be used instead of STR’s as they only need small DNA fragments of 40-50 bp.

Other advantages of SNP’s pointed out by him:

- 50-100 SNP`s for forensic casework needed

- High potential for automation

- Low mutation rate is attractive for parentage testing

- Deceased father but close relatives are available

Future Technologies and Improvements

µTAS-DNA Sequencing23

What about automated single cell isolation, cell lysis, restriction enzyme action, PCR, primer hybridization on a microarray with automatic detection? And all of this on an automated chip? Not really future technologies anymore as all of these components already exist in miniature form.

Studies have been and are being performed on forensic labs on chips but only for the separation and detection of the DNA.

Databases2

Future improvements for the current databases could be:

- Include molecular distance with haplotype diversity.

- Increase database samples.

- Centralised database for SNP markers.

Multiplex Kits

What do we need?

- New assays

- Evaluation of new STR’s

- Evaluate SNP methods

- Introduce more Standards

- According to Sanchez19, the development of SNP multiplexes for an initial screening for the major populations and the development of a large multiplex package that include Y-SNP`s that can discriminate between individual lineages in all populations is something which needs to be developed.

- Development of a single multiplex assay to amplify these SNP’s.

- Automation of the assays for high throughput.

- Research on different microarray technologies for the typing of these assays.

Conclusion

There are three things the community needs: kits, databases and experience.

Multiplex kits to improve the power of discrimination, databases to calculate the match probability in a suitable manner and experience with these new technologies.

I’m convinced that all of these will be available, as researchers are very keen to explore such an exciting field of genetics.

References

1. Profiles in DNA Available at:

[Accessed on 22nd October 2004]

2. IV. International Y-User Workshop ‘Haploid DNA Markers in Forensic Genetics’ 18th – 20th November 2004 in Berlin Germany

3. Kayser, M. et al. (2004) A comprehensive survey of Human Y-Chromosomal Microsatellites. Am. J. Human Genetics Vol. 74, pp. 1183-1197

4. Schoske, R. et al. (2004) High throughput STR typing of U.S. populations with 27 regions of the Y-chromosome using two multiplex PCR Assays. Forensic Science International Vol. 139, pp. 107-121

5. Pascali et al. (1998) Coordinating Y-chromosomal STR research for the courts. International Journal Legal Medicine Vol. 112, nr. 1, pp. 1

6. YHRD STR Database Available at:

[Accessed on 22nd October 2004]

7. Short Tandem Repeat DNA Internet Database. Available at:

[Accessed on 21st October 2004]

8. Butler, J. et al. (2002) A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Science International Vol. 129, pp. 10-29

9. Hanson, E. and Ballantyne, J. (2004) A highly discriminating 21 locus Y-STR "Megaplex" system designed to augment the minimal haplotype loci for forensic casework. Journal of Forensic Science Vol. 49, nr. 1

10. Daniels, D.L., Hall, A.M. and Ballantyne, J. (2004). SWGDAM Developmental Validation of a 19-Locus Y-STR System for Forensic Casework. Journal of Forensic Science Vol. 49, nr. 4

11. Hall, A. & Ballantyne, J. (2003) The Development of an 18 Loci Y-STR system for Forensic Casework. Analytical and Bioanalytical Chemistry Vol. 376, nr. 8, pp. 1234-1246

12. Innovative Y-STR Technology Available at:

[Accessed on 22nd October 2004)

13. AmpFlSTR® Y-filer™ PCR Amplification Kit Product Bulletin Available at:

[Accessed on 1st November 2004]

14. Die Mentype® Produktserie Available at:

[Accessed on 19th November 2004]

15. GeneKin® Y-STR multiplex systems Available at:

[Accessed on 19th November 2004]

16. Underhill, P.A., et al. (1997) Detection of numerous Y chromosome biallelic polymorphisms by denaturing High-Performance Liquid Chromatography. Genome Research Vol. 7, pp. 996-1005

17. Signet™ Y-SNP Identifcation System Available at:

[Accessed at 23rd October 2004]

18. Vallone, P. & Butler, J. (2004) Multiplexed assays for evaluation of Y-SNP markers in US populations. International Congress Series Vol. 1261, pp. 85– 87

19. Sanchez, J. J. et al. (2003) A Y-Chromosome SNP Assay for Co-amplification of 25 DNA Fragments and Simultaneous Detection of 35 Human Y-SNP’s. Forensic Science. International Vol 137, nr. 1, pp. 74-84

20. Inagaki S, Yamamoto Y, Doi Y, Takata T, Ishikawa T, Yoshitome K, et al. (2002)

Typing of Y chromosome single nucleotide polymorphisms in a Japanese

population by a multiplexed single nucleotide primer extension reaction.

International Journal of Legal Medicine Vol. 4, pp. 202–206

21. Ybase: Genealogy by numbers Available at:

[Accessed on 24th October 2004]

22. EMPOP - EDNAP mtDNA Population Database

[Accessed on 20th November 2004]

23. Royal Society of Chemistry Journals: Lab on a Chip Available at:

[Accessed on 15th October 2004]

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