# Presentation: 090142

## Y Chromosome DNA Variation Monitored by SNP and STR Analysis

- John M. Butler
**NIST Biotechnology Division**
**CHI 4th Annual DNA Forensics Meeting**
**June 1-2, 2000 (Springfield, VA)**

## Presentation Outline

- Advantages of Y Chromosome Markers
- Y Chromosome Structure and Variation
- Commonly Used Y Chromosome Markers
- Y STR Multiplex Assays
- Population Studies
- Y SNP Markers
- Y Chromosome Work at NIST

## Why the Y Chromosome?

- Applications
- forensic investigations **(98% of violent crime by men)**
- genealogical purposes
**evolutionary studies**
- Advantages to Human Identity Testing
- male component isolated without differential extraction
- paternal lineages
- Needs
- population studies to evaluate diversity of haplotypes
- robust assay for accurate characterization of Y markers

## Y Chromosome Structure

***Nucleic Acids Res.***** 28(2), e8 (2000)**

- ~60 Mb total DNA sequence (only chromosome 22 is smaller)

- p

- q

- 35-36 Mb euchromatin 9.5 Mb sequenced (27%)

**AMEL**

**SRY**

## Y Chromosome DNA Markers

- STRs (microsatellites)
- DYS19, DYS385, etc.
- mostly tetranucleotide repeats
- Bi-allelic markers (unique event polymorphisms--UEP)
- SNPs (single nucleotide polymorphisms)
- _Y_ _*A*_*lu* _p_olymorphism (YAP) or other insertions/deletions *(“indels”)*
- Minisatellite
- MSY1 (DYF155S1) composed of 48-114 copies of a 25 bp repeat unit with 5 sequence variant repeat types
- typed by MVR-PCR (minisatellite variant repeat)

## Y Chromosome STR Markers

- J.M. Butler, *Forensic DNA Typing,* Table 8.1

## New Y STR Markers

**Genomics 57, 433-437 (1999)**

- Y-GATA-A4

- Y-GATA-A7.2

- Y-GATA-H4

- Y-GATA-C4

- Y-GATA-A10

- Y-GATA-A7.1

- Y-GATA-A8

**New Y STR Markers**

## New Y STR Markers

**New Y STR Markers**

***Nucleic Acids Res.***** 28(2), e8 (2000)**

- DYS434

- DYS435

- DYS437

- DYS439

- DYS438

- DYS436

## Map of Y Chromosome STR Markers

***Nucleic Acids Res.***** 28(2), e8 (2000)**

- p

- q

## Current Forensic STR Multiplexes

- D13

- D3

- A

- FGA

- vWA

- D8

- D21

- D18

- D5

- D7

**Profiler PlusTM**

## Current Forensic STR Multiplexes

- D13

- D3

- D21

- TH01

- D5

- D7

**PowerPlexTM 16**

- D16

- D18

- Penta E

- A

- vWA

- D8

- TPOX

- CSF

- Penta D

- FGA

## Y STR Multiplex Assay

- DYS19

- 389II

- 389I

- 390

- _Primer_   _Amounts__	__Dye_
- Y19	0.25 M	JOE
- Y389	0.125 M	FAM
- Y390	0.25 M	JOE

*Prinz et al. 1997            **(Forensic Sci Int, vol. 85, pp. 209-218)*

**“****Quadruplex I”**

## Y STR Multiplex Assay

- 391

- 392

- 393

*Kayser et al. 1997               **(Int J Legal Med, vol. 110, pp. 125-133)*

- _Primer_   _Amounts__	__Dye_
- Y391	0.3 M	FAM
- Y392	0.3 M	JOE
- Y393	0.06 M	FAM

**“****Triplex I”**

## Y STR Multiplex Assay

*Ishii et al. 1999                            **(Progress in Forensic Genetics 8, pp. 457-459)*

- _Primer_   _Amounts__	__Dye_
- Y391	0.50 M	FAM
- Y392	0.30 M	JOE
- Y393	0.06 M	FAM
- Y19	0.20 M	NED
- Y385	0.05 M	FAM

**Modified “Triplex I”**

## Y STR Multiplex Assay

- 390

- 391

- 393

- _Primer_   _Amounts_
- Y19	 0.32 M
- Y390	 0.16 M
- Y391	 0.16 M
- Y393	 0.10 M

*Redd et al. 1997               **(Biol. Chem. vol. 378, pp. 923-927)*

**4-plex**

*all loci are FAM-labeled*

- 19

## Y STR Multiplex Assay

- DYS19

- 389II

- 389I

- 390

- _Primer_   _Amounts__	__Dye_
- Y19	0.4 M	TET
- Y389	0.12 M	TET
- Y390	0.12 M	FAM
- Y393	0.2 M	HEX

*Gusmão et al. 1999            **(Forensic Sci Int, vol. 106, pp. 163-172)*

**5-plex**

- 393

## Primer Sequences

*From ****Y STR Database***
*http://ystr.charite.de/*

## Y STR Multiplex Assay

*Thomas et al. 1999            **(Hum. Genet., vol. 105, pp. 577-581)*

## Y STR Multiplex Assay

- 389I

- 425

- _Primer_    _Amounts__	__Dye_
- Y388	0.185 M	TET
- Y389	0.350 M	TET
- Y425	0.150 M	FAM
- Y426	0.060 M	HEX

*Thomas et al. 1999            **(Hum. Genet., vol. 105, pp. 577-581)*

**“****MS2”**

- 388

- 426

- 389II

## Y STR Multiplex Assay

- 434

- 435

- _Primer_   _Amounts__	__Dye_
- Y434 	0.2 M	TET
- Y435 	0.05 M	TET
- Y436 	0.025 M	FAM
- Y437 	0.1 M	HEX
- Y438 	0.2 M	HEX
- Y439 	0.2 M	TET

- 436

- 438

- 437

- 439

*Ayub et al. 2000       **(Nucleic Acids Res., vol. 28, e8)*

**6-plex**

## “State of the Y STR Assay” 

- A number of multiplex reactions have been reported in the literature but Y STR multiplexes have not reached their potential...
- Very little PCR optimization to-date (most work has been done with the original PCR primer sequences)
- No commercial Y STR kit exists yet (therefore these markers remain inaccessible to the general forensic DNA community)
- New Y STR markers are becoming available which will greatly improve the power of discrimination between unrelated individuals (e.g., DYS385) and these will need to be incorporated into future multiplex sets

## Italian Population Haplotypes

*216 Unrelated Males*

- Y19

- Y389I

- Y390

- Y389II

- Y391

- Y392

- Y393

- Y385

*From Forensic Haemogenetic Laboratory *
***Catholic University of the Sacred Heart UCSC - ROME**** *
*http://www.mclink.it/personal/MD1696/data/*

## Italian Population

*From Forensic Haemogenetic Laboratory *
***Catholic University of the Sacred Heart UCSC - ROME**** *
*http://www.mclink.it/personal/MD1696/data/*

*216 Unrelated Males*

## DYS385 Allele Frequencies

- 11-14

- 13-17

**Allele Calls**

- 10-11

- 19-19

**45 different “alleles”**

*216 Unrelated Italian Males*

## # Haplotypes vs. # Males Tested

- Dupuy, B.M., et al. (2000) Progress in Forensic Genetics 8, pp. 260-262
- Ricci, U., et al. (2000) Progress in Forensic Genetics 8, pp. 275-277
- Henke, L., et al. (2000) Progress in Forensic Genetics 8, pp. 296-298
- Kupiec, T., et al. (2000) Progress in Forensic Genetics 8, pp. 312-314
- Mukoyama, R., et al. (2000) Progress in Forensic Genetics 8, pp. 318-320
- Gusmao, L., et al. (2000) Progress in Forensic Genetics 8, pp. 324-326
- Carvalho, M., et al. (2000) Progress in Forensic Genetics 8, pp. 302-304
- Aler, M., et al. (2000) Progress in Forensic Genetics 8, pp. 305-308

- 18th Congress of the International Society of Forensic Haemogenetics (ISFH), San Francisco 1999

## Y-STR Haplotype Reference Database

**Y-STR Haplotype Reference Database**

**http://ystr.charite.de/**

- +YCAII

## Summary of Y DNA Population Variation

- Fairly significant discrimination powers can be achieved when using many Y STR markers...very dependent on the population samples selected
- Population sub-structure exists and is more significant for Y SNPs
- We will need larger databases of Y STRs and Y SNPs for calculating powers of discrimination for Y haplotypes (for the same reasons as mtDNA)

## Y Bi-allelic Markers

- 154 Y SNPs from Peter Underhill (Stanford) discovered by DHPLC

## Y Haplotypes with Bi-allelic Markers

- Su, B., *et al*. (1999) *Am. J. Hum. Genet*. 65: 1718-1724

- S = small alleles
- L = large alleles
- W = wild-type alleles
- D = deletion alleles

**M15-M5-DYS287-M3-M119-M7-M50-M110-M89-M122-M88-M103-M45-M95-M9-M120-M111-M134-M17**

**17** different haplotypes

- 19 markers

**925 male DNA samples (739 from eastern-Asia)**

## Multiplex Restriction Enzyme Digestion

- 92R7, Tat, sY81, SRY+465, SRY4064, YAP

## Multiplex Restriction DigestionsM.G. Thomas et al. 1999            (Hum. Genet., vol. 105, pp. 577-581)

**“****UEP1” (6 Y SNPs assayed)**
_**Locus**__**	**__**	**__**Enzyme**__**	**__**PCR Size**__**	**__**Labeled size (cut)**_
- 92R7		*Hin*dIII	  55 bp		  28 (C)	  53 (T)
- Tat		*Nla*III		112 bp		  83 (T)	112 (C)
- sY81		*Nla*III		142 bp		105 (A)	142 (G)
- SRY+465	*Fnu*4HI	148 bp		  98 (C)	148 (T)
- SRY4064	*Bsr*BI		225 bp		135 (G)	174 (A)
- YAP		n/a		99/413 bp	  99 (-)		413 (+)

**R**
**R**
**F**
**R**
**F**
**     ****F**

## Multiplex Restriction DigestionsM.G. Thomas et al. 1999            (Hum. Genet., vol. 105, pp. 577-581)

**“****UEP2” (5 Y SNPs assayed)**
_**Locus**__**	**__**	**__**Enzyme**__**	**__**PCR Size**__**	**__**Labeled size (cut)**_
- M9		*Hin*fI		214 bp		  48 (C)	  76 (G)
- M13		*Bsp*143I	119 bp		  56 (G)	119 (C)
- M17		*Afl*III		124 bp		101 (.G)          124 (+G)
- M20		*Sxp*I		106 bp		  62 (A)	102 (G)
- SRY10831	*Dra*III		  73 bp		  41 (G)	  73 (A)

## NIJ-Funded RESEARCH EFFORT at NIST

**TECHNOLOGY**

**BIOLOGY**

**GENETICS**

- Capillary Electrophoresis

- MALDI-TOF
- Mass Spectrometry

- mtDNA

*John Butler*
*Pete Vallone*
*Christian Ruitberg*
*Dennis Reeder*

*Multiplex PCR*

*Automation*

*Primer Design Improvements*

*STRBase*

## NIST Work with Y Markers

- Improvement in Y STR Multiplex Assays
- Development of Y STR Standard Reference Material (haplotypes for SRM2391a components)
- Multiplex Y SNP Assay Development and Marker Evaluation
- Rapid Construction of Y Haplotype Databases Using High-throughput Capabilities of Time-of-Flight Mass Spectrometry

## Y STR Multiplex Assays

- primers

- Unlabeled amplicons with intercalating dyes (single-color)

*Rapid CE Separation*

*Capillary electrophoresis used for rapid separation and quantitation to adjust balance between loci and achieve robust multiplexes*

## Male-Specific Multiplex PCR at 17 Y SNP Loci

- 17 plex PCR

**Female sample (K562)**

**Male sample (AM209)**

- 20 bp ladder

- 200 bp

- 300 bp

- 400 bp

- CE Separation Time (min)

**Remaining primers**

## Less variation with Y STRs

- 9 repeats

- 20 repeats

- 16 repeats

- 30 repeats

- 8 repeats

- 15 repeats

- 8 repeats

- 12 repeats

**88 samples tested at 4 different STRs via mass spec**

## For More Information...

- Peter de Knijff’s Y STR web page:
- http://ruly70.medfac.leidenuniv.nl/~fldo/hptekst.html
- Y STR Haplotype database:
**http://ystr.charite.de**
- STRBase
**http://www.cstl.nist.gov/biotech/strbase**

## URL: www.cstl.nist.gov/biotech/strbase/

**URL: www.cstl.nist.gov/biotech/strbase/**

**Y chromosome section contains 136 references**

**Currently lists 1259 references for application of STRs to forensics**

**100 variant alleles reported for 13 CODIS STRs**

## Acknowledgments

- Christian Ruitberg
- Peter Vallone
- Dennis Reeder
- Margaret Kline
- Lisa Forman (NIJ)
- Peter Underhill (Stanford Univ.)

*Research partially funded by NIJ Grant 97-VX-LB-0003*