Use of Alternative Primers for Gender Discrimination in Human Forensic Genotyping

Doris M. KupferMarita JenkinsDennis Burian Dennis V. Canfield Civil Aerospace Medical InstituteOklahoma City, OK 73125

April 2008

Final Report

DOT/FAA/AM-08/8Office of Aerospace MedicineWashington, DC 20591

OK-08-1997

NOTICE

This document is disseminated under the sponsorship of the U.S. Department of Transportation in the interest

of information exchange. The United States Government assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine technical reports are available in full-text from the Civil Aerospace Medical Institute’s publications Web site:

www.faa.gov/library/reports/medical/oamtechreports/index.cfm

�

Technical Report Documentation Page 1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-08/8 4. Title and Subtitle 5. Report Date

April 2008 Use of Alternative Primers for Gender Discrimination in Human Forensic Genotyping 6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No. Kupfer DM, Jenkins M, Burian D, Canfield DV

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) FAA Civil Aerospace Medical Institute P.O. Box 25082 11. Contract or Grant No. Oklahoma City, OK 73125

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered Office of Aerospace Medicine Federal Aviation Administration 800 Independence Ave., S.W. Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes Work was accomplished under approved task AM-TOXLAB 16. Abstract An assay using the Federal Bureau of Investigation’s human Combined DNA Identity System (CODIS) primers has been developed for polymerase chain reaction (PCR)-based human identity testing. Recent forensic literature has identified several human populations that carry a deletion mutation in the Y-chromosome copy of the amelogenin locus. This is the standard locus used for gender determination in CODIS. Additionally, the amelogenin male PCR products are very close in size requiring manual annotation of PCR electrophoresis results for this locus. This study was designed to test several gender-specific primers which are to loci outside the amelogenin region, have well-separated PCR products, and could serve as additions or replacements to amelogenin in our human identity testing assay.

17. Key Words 18. Distribution Statement

PCR, Genotyping, Gender Determination, Electrophoresis Document is available to the public through the Defense Technical Information Center, Ft. Belvior, VA 22060; and the National Technical Information Service, Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified 10

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

���

ACkNOwlEdgmENT

We thank Dr. Brandt Cass�dy, DNA Solut�ons, Inc., for h�s helpful d�scuss�ons and for

prov�d�ng the �n�t�al ZFX/ZFY pr�mer m�xture for gender determ�nat�on.

v

CONTENTs

INtroDuCtIoN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

MAterIAlS AND MethoDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Sample sets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Genom�c DNA extract�on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

PCr pr�mers and ampl�ficat�on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

electrophores�s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

reSultS AND DISCuSSIoN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

reFereNCeS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1

Use of AlternAtive Primers for Gender discriminAtion in HUmAn forensic GenotyPinG

INTrOduCTION

Polymerase cha�n react�on-based human �dent�ty test-�ng has been used successfully for genotyp�ng of forens�c samples. the Federal Bureau of Invest�gat�on developed a standard�zed set of loc� and pr�mers for use w�th PCr for genotyp�ng. the Comb�ned DNA Ident�ty System (CoDIS) conta�ns a core set of Str markers for hu-man �dent�ty test�ng1 that are d�scr�m�natory over a w�de range of ethn�c�t�es. An add�t�onal marker, amelogen�n,2 has been �ncluded �n the CoDIS ser�es for gender d�s-cr�m�nat�on.

the amelogen�n pr�mers are spec�fic for a locus w�th�n the X-Y homologous reg�on. Gender determ�nat�on �s based on the presence of an X copy and the presence or absence of a 6 base-pa�r shorter Y copy.2 In 1998, Santos reported that two Sr� lankan �nd�v�duals from 350 males from var�ous ethn�c groups carr�ed a p-arm delet�on of the Y chromosome wh�ch �ncluded the amelogen�n re-g�on.3 thangaraj, �n a 2002 study of 270 Ind�an males, found five carry�ng a s�m�lar delet�on.4 In a 2003 study of 338 �nd�v�duals of Malay, Ch�nese, and Ind�an eth-n�c�ty Chang reported that four Ind�ans and one Malay showed a delet�on of the Y copy of the amelogen�n locus.5 there are add�t�onal reports of amelogen�n Y nulls �n an Austral�an Caucas�an,6 a Moroccan father-son pa�r,7 and s�x Austr�ans.8 Chang concludes that there appears to be an amelogen�n fa�lure as h�gh as 3.6% �n the Malays�an Ind�an populat�on and a lower but not�ceable defic�t of 0.02% �n the Caucas�an populat�on.5

recently, we reported the development of a genotyp�ng protocol based on the use of pr�mers from the CoDIS set, �nclud�ng the gender determ�n�ng marker, amelogen�n,9 us�ng m�croflu�d�cs rather than cap�llary electrophores�s for ampl�con detect�on. Based on the low but s�gn�ficant probab�l�ty of m�s�dent�fy�ng samples, we cons�dered �t advantageous to our genotyp�ng protocol to develop an add�t�onal gender determ�nat�on assay for use �n suspected Y nulls. Add�t�onally, we were �nterested �n develop�ng an assay that could potent�ally replace or be an addendum to the amelogen�n assay s�nce the very close s�ze d�ffer-ence between the X and Y allele products was somet�mes d�fficult for m�croflu�d�cs �nstruments to automat�cally detect, thus requ�r�ng manual annotat�on.

Add�t�onal genotyp�ng assays have been developed for a ser�es of Y-spec�fic markers, wh�ch �nclude a ser�es of short tandem repeat reg�ons �n the DYS locus found on the q-arm of the Y chromosome (see, for example, stud�es done by Butler10 and ru�tberg11). G�lson reported the use of a m�xture of pr�mers for two loc� that can be used �n a w�de var�ety of mammals �nclud�ng humans for sex determ�nat�on.12 one pr�mer pa�r �s des�gned to a reg�on of the Y-spec�fic gene SrY,12,13 and the second �s to the homologous z�nc finger prote�n genes of the X and Y chromosomes, ZFX/ZFY.14 the successful use of these pr�mers �n humans has been reported.15

the current study took advantage of the ava�lab�l�ty of human forens�c samples to test alternat�ve gender deter-m�n�ng pr�mers. We tested Y-spec�fic short tandem repeat pr�mer sets for loc� DYS390,10 DYS438, and DYS439,16 all q-arm loc�, and the pr�mers for p-arm loc� ZFX/ZFY and SrY. the results were compared to amelogen�n for confirmat�on of gender.

mATErIAls ANd mEThOds

Sample setsFour samples were used for character�zat�on stud-

�es. two male samples, 04010722-1 from l�ver, and 03010577-11 from blood, were forens�c �n or�g�n. Samples 23A and 23B were blood from female volunteers, col-lected under a local Internal rev�ew Board protocol for developmental work. After �n�t�al character�zat�on of the pr�mers, 12 add�t�onal forens�c samples were used to val�date the selected cond�t�ons across a var�ety of t�ssue types and DNA qual�ty (table 1).

genomic dNA extractionWe stored and processed samples as descr�bed �n a

prev�ous report.9 Br�efly, t�ssue and blood were stored at -20° C. Approx�mately 25 mg of t�ssue was m�nced and processed w�th the ChargeSw�tch gDNA M�n� t�s-sue k�t, #CS11204, us�ng the manufacturer’s d�rect�ons (Inv�trogen; Carlsbad, CA). DNA from whole blood was extracted from 200ul whole blood us�ng the QIAamp DNA M�n� K�t #51304 (Q�agen, Valenc�a, CA).

2

PCr primers and amplificationthe sequences for and expected product s�ze of the s�x

pr�mer pa�rs used �n the study are shown �n table 2. All pr�mers were synthes�zed commerc�ally (Integrated DNA technolog�es; Coralv�lle, IA). A m�x of ZFX/ZFY and SrY pr�mers for �n�t�al test�ng was prov�ded by Brandt Cass�dy, DNA Solut�ons; oklahoma C�ty, oK.

react�on setup was for 25ul final volume. each react�on conta�ned 5ng template, 400-800nM pr�mers, 2.5mM dNtPs, 0.1% tr�tonX-100, 1x Ampl�taq Gold buffer, and 2.25un�ts Ampl�taq gold. Ampl�ficat�on was as prev�ously reported.9 thermal cycl�ng was performed on a GeneAmp 9600 thermal cycler (Perk�nelmer; Wellesley, MA) us�ng the follow�ng cycl�ng profile for 40 cycles:

An �n�t�al 95° C 11 m�n, 96° C 1 m�n; then 94° C 30sec, ramp 68 sec to 60° C, hold for 30 sec, ramp 50 sec to 70° C, hold for 45 sec, for 10 cycles. then 90° C 30 sec, ramp 50 sec to 70° C, hold for 45 sec, for 30 cycles. then 60° C for 30 m�n, 4° soak.

ElectrophoresisDetect�on of a 1ul al�quot from a 1:3 d�lut�on �n d�st�lled

water of each ampl�ficat�on product was performed on a 2100 B�oanalyzer us�ng Ag�lent expert software vers�on B.02.03.SI307 and DNA 1000 Ser�es II labch�p k�ts #5067-1504 (Ag�lent technolog�es; Palo Alto, CA).

rEsulTs ANd dIsCussION

PCr was performed w�th the four character�zat�on samples us�ng DYS pr�mers at 800nM and m�xture pr�mers at 400nM under standard PCr cond�t�ons (see Mater�als and Methods sect�on). DYS390 and DYS439 gave the expected product for the two males and were negat�ve for female samples. DYS438 showed the expected s�ngle peak w�th the male samples but showed three products w�th the female. Due to the presence of these spur�ous products, DYS438 was el�m�nated from further test�ng. A m�xture of ZFX/ZFY and SrY pr�mers rece�ved from Brandt Cass�dy was tested at 400nM w�th the four samples and gave the expected product results, a s�ngle peak for females and two peaks for males (see table 2 for expected products and s�zes).

the cr�ter�a for a rel�able gender determ�nat�on test were the ab�l�ty to detect both males and females w�th a d�fferent�al s�gnal for each, as seen w�th amelogen�n.

Table 1. Additional forensic samples used in the study.

Sample ID Source 03010577-1 blood 03010577-7 liver 03010577-11 blood 08010522-1 blood 08010522-3 heart 08010522-5 brain 01010622-1 liver 01010622-3 kidney 04010622-4 kidney 08010622-1 blood 04010722-1 kidney 04010722-2 skin

Table 2. Primers used in study.

Locus Primer Sequence Product Size or

Range (bp) Gender Reference Amelogenin ACC TCA TCC TGG GCA CCC TGG TT 212(Y) or 218(X) X and Y Mannucci (2)

AGG CTT GAG GCC AAC CAT CAG

DYS390 TAT ATT TTA CAC ATT TTT GGG CC 189-233 Y-specific Butler (10)

GTG ACA GTA AAA TGA AAA CAT TGC

DYS438 TGG GGA ATA GTT GAA CGG TAA 202-242 Y-specific Ayub (16)

GTG GCA GAC GCC TAT AAT CC

DYS439 TCC TGA ATG GTA CTT CCT AGG TTT 236-256 Y-specific Ayub (16)

GCC TGG CTT GGA ATT CTT TT

SRY CCC ATG AAC GCA TTC ATT GTG TGG 224 Y-specific Gilson (12)

ATT TTA GCC TTC CGA CGA GGT CGA TA

ZFX/ZFY GCA CTT CTT TGG TAT CTG AGA AAG T 445 X and Y Aasen (14)

ATA ATC ACA TGG AGA GCC ACA AGC T

3

th�s ruled out the use of the DYS or SrY pr�mers, alone, s�nce they are Y-spec�fic. therefore, three pr�mer m�xes, each conta�n�ng the ZFX/ZFY pr�mer pa�r, wh�ch g�ves a character�st�c 445 bp product w�th a female sample, and one male-spec�fic pr�mer pa�r were tested. the results were compared to amelogen�n, wh�ch g�ves a s�ngle peak for a female template, a doublet peak w�th a male. the m�xes, all at 800nM for each pr�mer, were DYS390 + ZFX/ZFY, DYS439 + ZFX/ZFY, and SrY + ZFX/ZFY. the last m�x, SrY+ZFX/ZFY, �s s�m�lar to the one rece�ved from Brandt Cass�dy but at 800nM for each pr�mer, rather than 400nM (F�gure 1).

From F�gure 1A, �t �s clear that the small d�fference �n s�ze of the X versus Y chromosome amelogen�n product results �n a doublet rather than the well-separated peaks as seen �n F�gure 1B-D for male samples w�th the three pr�mer m�xes. Note that the ZFX/ZFY products seen for the female sample have a h�gher s�gnal than the male s�nce there are two cop�es of the X chromosome present. Also note that secondary products are present for the male samples for all Y-spec�fic pr�mers tested, �nclud-�ng amelogen�n. All three m�xes gave d�st�nct peaks for male samples. there was, however, a d�fferent�al level of s�gnal between the three Y-spec�fic pr�mer products and ZFX/ZFY. ZFX/ZFY showed a fluorescent un�t (Fu) measure on the B�oanalyzer of roughly 100 Fu at 800nM w�th the male samples. But the Y-spec�fic pr�m-ers ranged from a h�gh of 100 Fu w�th SrY to 40 Fu w�th DYS390 to a low of less than 40 Fu w�th DYS 439 (F�gure 1B-D). the ampl�tude of secondary products w�th DYS and SrY pr�mers were all �n the range of 5-15 Fu. the SrY + ZFX/ZFY m�x was determ�ned to have opt�mal performance of the three pr�mer m�xes show�ng the h�ghest overall s�gnal �ntens�t�es of products, the most closely equ�valent product s�gnal �ntens�t�es, and lowest secondary product s�gnal strengths.

We attempted to reduce the secondary products presence by reduc�ng the pr�mer concentrat�on �n the SrY + ZFX/ZFY m�x. A range of 400, 600, and 800nM was exam�ned for product and secondary product peak strength w�th the same four samples. No secondary products were detected at 400nM, but s�gnal �ntens�ty was reduced to 15 Fu. Increas�ng SrY concentrat�on to 600 and 800nM, respect�vely, showed the presence of the secondary product doublet at less than 5 Fu for 600nM and 5-8 Fu 800nM. the SrY ampl�con peak he�ght at 600nM averaged 50 Fu and 70 Fu at 800nM (F�gure 2). the relat�vely low level of secondary prod-uct and h�ghest product Fu at 800nM suggested that th�s was the opt�mal concentrat�on. the d�fference �n

Ameloginin

Low MW Marker

High MW Marker

Secondary products

A.

Low MW Marker

High MW Marker

SRY

DYS390

DYS439

ZFX/ZFY

Secondary products

B.

C.

D.

Low MW Marker

High MW Marker

Secondary products

Low MW Marker

High MW Marker

Secondary products

ZFX/ZFY

ZFX/ZFY

female

male

male male

female

male

male

female

male

female

male

Figure 1. Gender primer mix PCR products. Shown are overlays of bioanalyzer results of the PCR products from the three mixes and amelogenin (see Results and Discussion section). Male sample 04010722-1 and female sample 23A (taller peaks in Amelogenin and ZFX/ZFY overlays) are compared. A. Amelogenin; insert is a close-up of the characteristic male doublet. B. SRY+ZFX/SFY C. DYS390+ ZFX/SFY D. DYS439+ ZFX/SFY.

4

s�gnal �ntens�ty seen �n the two SrY PCr ser�es us�ng 800nM pr�mers w�th the same templates suggests that the var�at�ons between exper�ments may result �n s�gnals rang�ng from 70-100 Fu for the SrY products and 5-15 for the secondary product doublet. these levels are acceptable for detect�on on the B�oanalyzer �n the case of the product and rema�n below the default cutoff for the secondary products.

twelve forens�c samples were tested w�th the m�x at 800nM to assure cons�stent results across a var�ety of samples. these samples had been prev�ously tested by PCr w�th amelogen�n. All the samples appeared male, based on amelogen�n results, except 04010722-2, wh�ch gave a negat�ve amelogen�n result (data not shown). Four samples were from blood, and e�ght were from a var�ety of t�ssues (table 1). eleven of the samples showed the expected peaks for SrY and ZFX/ZFY at s�gnal strengths rang�ng from 50-100 Fu. Sample 04010722-2 showed a very weak s�gnal, a s�ngle peak for SrY w�th 10 Fu but no ZFX/ZFY peak. th�s was not surpr�s�ng s�nce th�s sample had been negat�ve w�th amelogen�n. the absence of the ZFX/ZFY peak, wh�ch represents an ampl�con of 445bp compared to the weak 245bp SrY ampl�con, sug-gests sample degradat�on. Furthermore, the presence of a peak from the SrY locus unamb�guously determ�nes that the sample �s from a male, regardless of the presence or absence of a s�gnal from the X-determ�nant locus. over-all, the gender m�x performs sat�sfactor�ly w�th forens�c samples, g�v�ng expected results w�th DNA �solated from both t�ssue and blood.

Figure 2. Comparison of PCR product signal strengths with SRY +ZFX/ZFY primer mixes. The product peaks are from male sample 04010722-1. The concentration of primers in the mix is shown.

400nM

600nM

800nMSRY

ZFX/ZFY

Secondary Products

400nM

600nM

800nM

In summary, the ava�lab�l�ty of add�t�onal gender determ�nat�on loc� w�ll expand our reperto�re of robust pr�mers for use �n forens�c sample �dent�ficat�on. th�s �s �mportant because of the null-Y mutat�on that results �n a delet�on of the amelogen�n gene located at 6.79Mbp on the Y chromosome.3 4 5 the mutat�on occurs espec�ally w�th�n certa�n Ind�an populat�ons but has also been found �n Caucas�an populat�ons.6 7 8 the result of th�s delet�on �s the �ncorrect �dent�ficat�on of the �nd�v�dual as female when us�ng amelogen�n as the gender-determ�n�ng locus. the extent of the mutat�on has been est�mated. the MSY1 locus, a Y-spec�fic m�n�-satell�te marker on the same p-arm as amelogen�n, but less than a Mbp away, was negat�ve w�th an amelogen�n delet�on group.5 Subjects carry�ng the null-Y marker had a Y-chromosome delet�on of about 1Mbp.4 F�nally, the tSPYP1 locus, located at 6.19Mbp, was found to be a prox�mal endpo�nt of the delet�on.3 the StY and ZFY genes are on the p-arm of the Y chromo-some at 2.56Mbp and 2.86 Mbp respect�vely. Both are s�gn�ficant d�stances from the amelogen�n gene delet�on reg�on and therefore are good cand�dates for alternat�ve gender determ�nat�on loc�.

Certa�nly, �n human forens�c cases where there �s a poss�b�l�ty of an amelogen�n delet�on, �t would be neces-sary to use an alternat�ve locus. however, �t also w�ll be very helpful to use the ZFX/ZFY and SrY pr�mer m�x to fac�l�tate automat�c annotat�on of the B�oanalyzer results because of the excellent separat�on and s�gnal strength of the products.

5

rEfErENCEs

1. Budowle B., Morett� t.r., N�ezgoda S.J., & Brown B.l. (1998). CoDIS and PCr-based short tandem repeat loc�: law enforcement tools. Proceedings of the Second European Symposium on Human Identi-fication; 1998; orlando, Fl; 73-88.

2. Mannucc� A., Sull�van K.M., Ivanov P.l., & G�ll P. (1994). Forens�c appl�cat�on of a rap�d and quant�tat�ve DNA sex test by ampl�f�cat�on of the X-Y homologous gene amelogen�n. International Journal of Legal Medicine 106, 190-3.

3. Santos F.r., Pandya A., & tyler-Sm�th C. (1998). rel�ab�l�ty of DNA-based sex tests. Nature Genetics 18, 103.

4. thangaraj K., reddy A.G., & S�ngh l. (2002). Is the amelogen�n gene rel�able for gender �dent�f�cat�on �n forens�c casework and prenatal d�agnos�s? Inter-national Journal of Legal Medicine 116, 121-3.

5. Chang Y., Burgoyne l.A., & Both K. (2003). h�gher fa�lures of amelogen�n sex test �n an Ind�an popula-t�on group. Journal of Forensic Science 48, 1-5.

6. roffey P.e., eckhoff C.I., & Kuhl J.h. (2000). A rare mutat�on �n the amelogen�n gene and �ts potent�al �nvest�gat�ve ram�f�cat�ons. Journal of Forensic Science 45, 1016-19.

7. henke J., henke l., Chatthopadhyay P., Kayser M., Dulmer M., Cleef S., Poche h., & Feske-Zech h. (2001). Appl�cat�on of Y-chromosomal Str haplotypes to forens�c genet�cs. Croatian Medical Journal 42, 292-7.

8. Ste�nlechner M., Berger B., & Ne�derstatter h. (2002). rare fa�lures �n the amelogen�n sex test. In-ternational Journal of Legal Medicine 116, 117-20.

9. Kupfer D.M., hugg�ns M., Cass�dy B., Vu N., Bur�an D., & Canf�eld D.V. (2006). A rap�d and �nexpens�ve PCr-based Str genotyp�ng method for �dent�fy�ng forens�c spec�mens. Wash�ngton DC: Office of Aerospace Medicine Report Dot/FAA/AM-06, 1-14.

10. Butler J.M., Schoske r., Vallone P.M., Kl�ne M.C., redd A.J., & hammer, M.F. (2002). A novel mult�plex for s�multaneous ampl�f�cat�on of 20 Y chromosome Str markers. Forensic Science International 129, 10-24.

11. ru�tberg M., reeder D.J., & Butler J.M. (2001). StrBase: A short tandem repeat DNA database for the human �dent�ty test�ng commun�ty. Nucleic Acids Research 29, 320-2.

12. G�lson A., Syvanen M., lev�ne K., & Banks J. (1998). Deer gender determ�nat�on by polymerase cha�n react�on: val�dat�on study and appl�cat�on to t�ssues, bloodsta�ns, and ha�r forens�c samples from Cal�forn�a. California Fish and Game 84, 159-69.

13. Fa�n S.r. & lemay P.J. (1995). Gender �dent�f�ca-t�on of humans and mammal�an w�ldl�fe spec�es from PCr ampl�f�ed sex-l�nked genes. Proceedings of the American Academy of Forensic Science Annual Meeting; 1995; Seattle, WA; 202.

14. Aasen e. & Medrano J.F. (1990). Ampl�f�cat�on of the ZFY and ZFX genes for sex �dent�f�cat�on �n humans, cattle, sheep and goats. Biotechniques 8, 1279-81.

15. McKeown B., St�ckley J., &r�ordan A. (2000). Gender ass�gnment by PCr of the SrY gene: an �mprovement on amelogen�n. Progress in Forensic Genetics 8, 433-5.

16. Ayub Q., Mohyudd�n A., Qamar r., Mazhar K., Zerjal t., Mehd� S.Q., & tyler-Sm�th C. (2000). Ident�f�cat�on and character�sat�on of novel human Y-chromosomal m�crosatell�tes from sequence da-tabase �nformat�on. Nucleic Acids Research 28, e8.