ELSEVIER SCIENCE IRELAND

Forensic Science International 65 (1994) 169-175

Forensic Science

international

.Analysis of short tandem repeat (STR) HUMVWA in the Spanish population

Jlose A. Lorente*a, Miguel Lorentea, Bruce Budowleb, Mark R. Wilsonb, Enrique Villanueva”

‘Department of Legal Medicine, University of Granada, Av. Madrid Il. 18012 Granada, Spain hFSRTC, Laboratory Division, FBI Academy, Quantico, Virginia 22135. USA

(Receiced 8 March 1993; revision received 17 November 1993; accepted 19 November 1993)

Abstract

Amplification by polymerase chain reaction (PCR) of variable number of tandem repeat (VNTR) loci and subsequent typing by electrophoresis and silver staining has become a useful tool for identity testing. One viable group of genetic markers amenable to amplification by PCR is the short tandem repeat (STR) loci. A horizontal discontinuous polyacrylamide gel electrophoresis (PAGE) method was used to type the amplified products of the STR HUMVWA. Typing for ‘VWA of 120 unrelated Spanish Caucasians was done. Six alleles were observed with frequencies in the range 0.096-0.242. The genotype distribution meets Hardy- Weinberg expectations (0.25 < P < 0.50). The heterozygosity was 73.3% and the discrimina- tion power (DP) 0.94. Simultaneously, in a small sample of families (n = 24) no new mutations could be bound.

Key words: PCR; VNTR; STR; Forensic identity; Population genetics

1. Introdulction

Since its introduction, DNA amplification by the polymerase chain reaction (PCR) [lj and subsequent electrophoresis of the amplified products has become a useful approach for typing variable number of tandem repeat (VNTR) loci [2,3].

The technique of PCR could be useful for paternity testing, as well as the forensic analysis of biological specimens, particularly where limited amount of sample or

* Corresponding author.

0379-0738/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 03791-0738(94)01474-6

170 J. A. Lorente et al. /Forensic Sri ht. 65 (1994) 169-I 75

highly degraded materials are the only sources of DNA [4-61. In recent years, various loci have been tested and determined valid for forensic purposes and one of these loci is HLA-DQa [7-91. An alternate group of genetic markers that are highly polymorphic are the VNTR loci. Loci such as DlS80 [lo], D17S5 [2] and ApoB [3] have been typed by amplification of DNA by PCR and subsequent electrophoresis of the amplified products (so-called AMP-FLPs) [ 11,121.

To enhance the discrimination power (DP) of AMP-FLP systems, additional loci are needed. Among the potential loci recently described is a class of AMP-FLPs known as short tandem repeats (STRs). The differences between successive alleles (i.e., tandem repeats) of STRs generally are 2-5 base pairs (bp) 112,131. Among other reported advantages, STRs have an approximately IO-fold higher sensitivity in comparison to AmpFLP systems [ 141, and the relatively short fragments are clear- ly associated with much higher success rate in severely degraded stains [ 151.

An STR locus called HUMVWA has been described. On chromosomal location 12p12-12pter of the von Willebrand (vWF) gene, intron 40 contains three different (TCTA) tandem repeat regions, two of them occurring between nucleotides 1900-2000, and between nucleotides 2200-2350; the third one (named VWA) is located between nucleotides 1640- 1794 [ 161. The present paper describes a simple analytical procedure for typing the alleles of HUMVWA and provides frequency data for VWA alleles from a Spanish sample population.

2. Materials and methods

Whole venous blood samples were drawn in ethylenediaminetetraacetic acid (EDTA) vacutainer tubes from 120 unrelated Spanish Caucasians living in An- dalucia (southern Spain). The DNA was isolated from the blood by organic extrac- tion, as described elsewhere [ 171, and quantified by slot-blot analysis using the probe D17Zl [18].

The primers were 5 ‘-CCCTAGTGGATGATAAGAATAATC-3 ’ and 5 ‘-GGA- CAGATGATAAATACATAGGATGGAT-3’ [19]. The components of the PCR were 5 ng of genomic DNA, 1 ~1 each dNTP (10 mM stocks), 5 ~1 10 x PCR buffer (Perkin-Elmer), 0.25 ~1 of each primer (from 100 PM stock solutions), and 2.5 units of Taq polymerase. The final volume was brought to 50 ~1 with sterile doubled distilled H,O.

The PCR was carried out in a Perkin-Elmer 9600 Thermal Cycler. The cycles were 94°C for 10 s for denaturing, primer annealing at 63°C for 10 s, and primer extension at 72°C for 10 s. The total number of cycles was 30.

After PCR the amplified alleles were separated in a discontinuous horizontal ultrathin-layer polyacrylamide gel (8% T, 5% C), cast on mylar sheets (GelBondTM, FMC, Rocklandn, ME, USA) by the flap technique [20]. GelBondTM backing facilitates manipulation of an ultrathin-layer gel through the staining process. The cross-linker was piperazine di-acrylamide [l I]. The gel buffer was 60 mM tris- formate, pH 9.0. The electrode buffer was 0. 28 M Tris-borate pH 9.0, containing a final concentration of 0.01% of bromophenol blue to serve as a dye marker for the moving boundary. Paper blotting strips (1 cm x 13.5 cm) (BRL, Gaithersburg, MD, USA) were soaked in the electrode buffer, excess buffer was removed by lightly blot-

J.A. Lorente ei al. /Forensic Sci ht. 65 (1994) 169-175 171

ting with paper towels, and the paper strips (two layers thick) were placed on the anodal and cathodal ends of the polyacrylamide gel - 20 cm apart from each other.

Five microliters of amplified product were applied to fiber glass applicator tabs (2.5 x 5 mm). The tabs were placed on the gel surface 1 cm from the cathode. An EC apparatus (EC Corporation, Saint Petersburg, FL) was used for electrophoresis. The electrophoresis power supply was set at 600 V, 20 mA, and maximum wattage. The temperature of the electrophoretic cooling platen was maintained at 15°C. The electrophoretic process was stopped when the moving boundary migrated to the anodal plulg.

The alleles were visualized using a silver staining procedure [l 11. Although not shown in Fig. 1, standard gels included a K562 positive control (Promega, Madison, WI, USA), a negative control, and both 10 bp and 123 bp ladders (BRL, Gaithesburg, MO, USA) to size the alleles.

3. Results and discussion

AMP-FLP typing requires a simple, high-resolution electrophoretic method for separating the particular alleles of interest and an adequate detection method. A high resolution, discontinuous buffer, horizontal polyacrylamide gel system can make AMP-FLP analysis easy and inexpensive [11,21-231. All alleles could be

Fig. 1. VWA gel picture. From left to right, 123L: 123 base-pair ladder. Lanes I-8 show the alleles from seven different individuals 1: 15,19; 2: 16.16; 3: 17,19; 4: 17,18; 5: l&17; 6: negative control; 7: 15,15; 8: 17.20. Individuals 6-8 conform to a paternity trio, where no exclusion could be found (6: child; 7: mother;

8: putative father). RAL: Reamplified allelic ladder (four lanes); AL: allelic ladder.

172 J.A. Lorente et al. /Forensic Sri ht. 65 (1994) 169-175

resolved with the technique described in this paper where a good resolution of alleles differing by one repeat unit may be observed (Fig. 1).

The electrophoretic system yields high resolution of PCR products by initial zone sharpening by means of a discontinuity in ionic strength between the sample and the gel [24]. An additional sequential zone sharpening of the partially separated DNA fragments occurs by a moving ion boundary generated by a discontinuous buffer sys- tem [23].

To facilitate allele typing, an allelic ladder for VWA was constructed. Since there are only six or seven relatively common alleles for VWA, a ladder was made by co- amplification of mixed heterozygous samples. Alternatively, individual alleles could be isolated, mixed together, and then subjected to PCR to construct an allelic ladder, following a procedure described elsewhere [25]. Once an allelic ladder was available, alleles were determined by comparison with the ladder, which was run on the same electrophoretic gel (Fig. 1). Bands shifts were never observed and, because of the good resolution between consecutive alleles, the system allowed unambiguous allele designation. Thus, typing of the samples was similar to the approach used for con- ventional protein electrophoretic genetic marker systems.

Allele frequencies for the VWA locus in the Spanish population are shown in Fig. 2 and Table 1. According to Kimpton, Walton and Gill [ 191 alleles were initially designated 1-7, but since alleles are now given a designation in terms of number of repeats, they have been redesignated nos. 14-20. To establish comparisons, allele no. 14 is the smallest in size and corresponds to the original allele no. 1, and the largest

% 30

SPANISH 0 BRITISH 25 .---- .._...._._ _____.

20 ....................

15 ......... .....

10

5 .........

0 14 15 16 17 18 19 20

allele number

Fig. 2. VWA allele frequencies from two Caucasian populations (Spanish, n = 120 individuals, and British, n = 100 individuals [19]).

J.A. Lmenre et al. /Forensic Sci Inr. 65 (1994) 169-175 173

Table 1 VWA allele frequencies in 120 unrelated Spanish Caucasians

Allele Frequency

I4 0.163 15 0.129 I6 0.208 I7 0.242 I8 0.163 I9 0.096 20 ---

one (no. 7) observed in the authors’ Spanish population sample is now called no. 20 (Kimpton and Gill, pers. commun.).

The auth.ors observed six out of seven alleles described in the original paper from a sample of British Caucasians [19]. Allele 7 was not observed in the Spanish popula- tion sample; however, it also was relatively rare (i.e., 0.01) in the British population study. The six alleles observed range in frequencies from 0.096 (allele 19) to 0.242

Table 2 VWA genotypes in 120 Spanish Caucasians

Genotype No.” observed No. expected X2

14.14 6 3.2 2.45 l4,15 3 5.0 0.80 14.16 7 8.1 0.15 14,17 II 9.5 0.24 14,18 3 6.4 I.81 l4,19 3 3.8 0.17 l5,lS 2 2.0 0.00 l5,16 8 6.4 0.40 15,17 7 7.5 0.03 15,18 5 5.0 0.00

15.19 4 3.0 0.33 16,16 7 5.2 0.62 l6,17 IO 12.1 0.36 16,18 7 8.1 0.15 16.19 4 4.8 0.13 17,17 IO 7.0 1.29 17,18 8 9.5 0.24 17,19 2 5.6 2.31 l8,18 5 3.2 1.01 l8,19 6 3.8 1.27 19.19 2 1.1 0.74 Total 120 120.3 14.50b

‘No. = number. b~2 = 14.50; 0.25 < P < 0.50; d.f. = 15

174 J.A. Lorenfe et al. /Forensic Sci Int. 65 (1994) 169-175

(allele 4). There were 21 genotypes observed in 120 unrelated Spaniards (Table 2). The DP, according to Fisher’s formula [26], was 0.94, and the observed heterozygosity was 73.3%. The genotype distribution for the Spanish sample popula- tion meets Hardy-Weinberg expectations (0.25 < P < 0.50) (Table 2). The Spanish data were compared with a British Caucasian sample population [ 191 using a x2 test for homogeneity (the program was kindly provided by G. Carmody, Carleton Uni- versity, Ottawa, Canada). The allele frequency distributions were similar (P = 0.311) (Fig. 2).

Simultaneously, a total of 24 families (54 meioses) have been tested using VWA. The segregations followed Mendelian inheritance and no mutations were observed using the described electrophoresis system.

In conclusion, a simple method for typing VWA is described that could be im- plemented into most laboratories at relatively low cost. In addition, a population database for VWA has been established for identity testing purposes. Further studies are underway to type other population samples, as well as identify other potential loci for identity testing. Furthermore, the usefulness of VWA in the analysis of fo- rensic and/or paternity cases is being evaluated.

4. Acknowledgements

This work was partially supported by a 1991 grant from the Scientific Committee of NATO to J.A. Lorente and by a 1992 grant from the Junta de Andalucia to M. Lorente.

5. References

2

3

8

9

IO

R. Saiki, S. Scharf, F. Faloona, K. Mullis, G.T. Horn and A. Erlich. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230 (1985) 1350-1354. G.T. Horn, B. Richards and K.W. Klinger, Amplification of a highly polymorphic VNTR by the polymerase chain recation. Nucleic Acid Res., I7 (1989) 2140. E. Boerwinkle, W. Xiong, E. Fourest, and L. Chan, Rapid typing of repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3’ hypervariable region. Proc. Nat. Acad. Sci. USA, 86 (1989) 212-216. A.J. Jeffreys, M.J. Allen, E. Hagelberg and A. Sonnberg, Identification of the skeletal remains of Joseph Mengele by DNA analysis. Forensic Sci. Inf., 56 (1992) 65-76. P. Gill, C.P. Kimpton, and K. Sullivan, A rapid PCR method for identifying fixed specimens. Elecfrophoresis, I3 (1992) I73- I75 E. Hagerlberg, I.C. Gray, A.J. Jeffreys, Identification of the skeletal remains of a murder victim by DNA analysis. Nature, 352 (1991) 427-429. R.K. Saiki, P.S. Walsh, C.H. Levenson and H.A. Erlich, Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Nat. Acad. Sci. USA, 86 (1989) 6230-6234. C.T. Comey and B. Budowle, Validation studies on the analysis of the HLA DQx locus using the polymerase chain reaction. J. Forensic Sci., 36 (1991) 1633-1648. C.T. Comey, B. Budowle, D.E. Adams, A.L. Baumstark, J.A. Lindsey and L.A. Presley, PCR am- plification and typing of the HLA DQx gene in forensic samples. J. Forensic Sci., (1993), in press. K. Kasai, Y. Nakamura and R. White, Amplification of a variable number of tandem repeats (VNTR) locus (pMCTI IS) by the polymerase chain reaction (PCR) and its application to forensic science. J. Forensic Sci., 35 (1990) 1196-1200.

J.A. Larente et al. /Forensic Sci Int. 65 (1994) 169-l 75 175

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

9. Budowle, R. Chakraborty, A.M. Giusti, A.J. Eisenberg and R.C. Allen, Analysis of the VNTR locus Dl!380 by the PCR followed by high-resolution PAGE. Am. J. Hum. Genet., 48 (1991) 137-144. A. Edwarsds, A. Civitello, H.A. Hammond and C.T. Caskey, DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet., 49 (1991) 746-756. M.H. PoJymeropoulos, D.S. Rath, H. Xiao and C.R. Merril, Tetranucleotide repeat polymorphism at the human beta-actin related pseudogene H-beta-AC-psi-2 (ACTBPZ). Nucleic Acids Res., 20 (1992) 14.32. S. Rand, NC. Puers, K. Skowasch, P. Wiegand, 9. Budowle, 9. Brinkmann, Population genetics and forensic efficiency data of 4 AMPFLPs. Inc. J. Leg. Med., 104 (1992) 329-333. P. Wiegand, 9. Budowle, S. Rand and 9. Brinkmann, Forensic validation of the STR systems SE33 and TCII.. Inr. J. Leg. Med., 105 (1993) 315-320. D.J. Mancuso, E.A. Tuley, LA Westfield, N.K. Worrall, 9.9. Shelton-lnloes, J.M. Sorace, Y.G. Alevy and J.E. Sadler, Structure of the gene for human von Willebrand factor. J. BioI. Chem., 264 (1989) 19514-19527. 9. Brinkmann, S. Rand and P. Wiegand, Population and family data of RFLP’s using selected single- and multi-locus systems. ht. J. Leg. Med., 104 (1991) 81-86. J.S. Waye, L.A. Presley, 9. Budowle, G.S. Shutler, and R.M. Fourney, A simple and sensitive meth- od for quantifying human genomic DNA in forensic specimen extracts. EioTechniques. 7 (1989) 852-855. C. Kimpton, A. Walton and P. Gill, A further tetranucleotide repeat polymorphism in the VWF gene. Hum. Molec. Genet., 1 (1992) 287. R.C. Allen, Rapid isoelectric focusing and detection of nanogram amounts of proteins from body tissues and fluids. Electrophoresis, 1 (1980) 32-37. 9. Budowle and R.C. Allen, Discontinuous polyacrylamide gel electrophoresis of DNA fragments. In C. Matthew (ed.), Methods in Molecular Biology, Vol. 9. Protocols in Human Molecular Genetics, Humana Press Inc., Clifton, New Jersey, 1991, pp. 123-132. R.C. Allen, Polyacrylamide gel electrophoresis with discontinuous voltage gradients. In Walter de Gruyter (ed.), Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, Berlin. 1971, pp. 29-32. R.C. Allen, G. Graves and 9. Budowle, Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver. BioTechniques, 7 (1989) 736-744. S. Hjerten, S. Jerstedt, and A. Tiselius, Some aspects of the use of continuous and discontinuous buffer systems in polyacrylamide gel electrophoresis. Anal. Biochem., 11 (1965) 219-223. F.S. Baechtel, J.B. Smerick, K.W. Presley and 9. Budowle, Multigenerational amplification of a reference ladder for alleles at locus DlS80. J. Forensic Sci., 35 (1993) 1176-I 182 R.A. Fisher, Standard calculations for evaluating a blood group system. Heredity, 5 (1951) 95-102.