Comparison of German population data on the apoB-HVR locus with other Caucasian, Asian and Black populations

Forensic Science International 80 (1996) 221-227

ELSEVIER

Forensic tiince Intemtii

Comparison of German population data on the apoB-HVR locus with other Caucasian, Asian and Black populations Roger P61tla’*, Christine Luckenbach”, Jiirgen Reinholdb, Rolf Fimmers”, Horst Ritter” “Institut

f iir Anthropologie

‘Institut

f

und Humangenetik, Wilhelmstrasse 27, 72074, Tiibingen, Germany hLabor Dr. Arnold, Hangerkirchgasse 7, 97070, Wiirzburg, Germany iir Medizinische Statistik, Universitiit Bonn, Sigmund Freud Strasse 25, 5310.5, Bonn, Germany

Received

29 June 1995; revised 1 February

1996; accepted 6 February

1996

Abstract A population study of 505 unrelated individuals from Southwestern Germany was carried out on the 3’-apoB hypervariable region (HVR). After amplification via polymerase chain reaction (PCR) and agarose gel electrophoresis, 15 different alleles and 47 genotypes were observed. The most common alleles were hypervariable elements (HVE) 37 and 3.5 with an allele frequency of 0.374 and 0.244, respectively. The heterozygosity index was calculated to be 78.4%. Allele frequencies of this study are compared with results from other databases obtained from a French, a Spanish, an Asian and an American (Black) population. Keywords: Hypervariable region; Apolipoprotein

B; Allele frequencies

1. Introduction The human apolipoprotein B (apoB) gene is located on the short arm of chromosome 2 (2p23-~24) and consists of 29 exons and 28 introns which span 43 kb [l]. It encodes both, apoB-48 and apoB-100 [2,3]. ApoB-100, having a *Corresponding

author, Tel.: +49 7071 296877; fax: +49 7071 296409.

0379-0738/96/$15.00 @ 1996 Elsevier PII SO379-0738(96)01917-Z

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molecular mass of 550 kDa is one of the largest proteins known so far and is present in VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins) and LDL (low density lipoproteins). ApoB-48 has a molecular mass of 260 kDa and is part of chylomicrons and chylomicron remnants. While apoB-100 is mainly synthesized in the liver, apoB-48 is produced in the small intestine. As apoB-100 functions as the ligand for the receptor-mediated uptake of LDL from serum into cells, it plays an important role in cholesterol homeostasis. In 1986 Knott et al. [4] sequenced a tandem repetitive region at the 3’ end of the human apoB gene. He found 11 closely related AT-rich repeat units, some containing a single dC or dG nucleotide instead of a dA. The HVEs were 11-16 bp in length. One year later, Huang and Breslow [5] detected different alleles at this locus as restriction fragment length polymorphisms (RFLPs). In 1989 Boerwinkle et al. [6] and Ludwig et al. [7] analyzed this minisatellite-, VNTR- (variable number of tandem repeats) or HVR- (hypervariable region) locus via the polymerase chain reaction (PCR) technique and showed the polymorphism and the variability of this region. Meanwhile, the 3’-apoB HVR has turned out to be a reliable genetic marker and is often applied to forensic and human genetic purposes. In this study we provide allele frequency data from a Southwestern German population and compare the data to previously published results.

2. Materials

and methods

2.1. DNA preparation

Genomic DNA was isolated from leucocytes according to Miller et al. [8]. 2.2. Oligonucleotides

Oligonucleotides were synthesized with the DNA synthesizer ‘Gene Assembler Plus’ (Pharmacia, Freiburg) and deprotected and desalted with NAP-10 columns (Pharmacia, Freiburg). 2.3. Polymerase chain reaction

The following PCR primers were used [7]:Primer 1: 5’-CAC AGC AAA ACC TCT AGA ACA-3’; Primer 2: 5’-GTT CCT CAG GAT CAA AGT ATG TAC-3’. DNA was amplified by three PCR-cycles at 94”C/2 min and 62”C/3 min and 28 PCR-cycles at 94YY1.5 min and 61”C/3 min on a Perkin Elmer 2400 PCR Thermocycler. PCR was carried out in a lOO-,~l reaction mixture containing 200 mM dATP,

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dCTP, dGTP and dlTP, 0.5 U Taq DNA polymerase, 1.5 mM MgCl,, 100 pmol of each primer and 50-70 ng DNA. 2.4. Agarose gel electrophoresis

and detection

PCR-fragments were separated in 2% agarose gels (0.8 X 20 X 20 cm; 60 V, 17 h; NuSieve 3:l agarose or Metaphor agarose -- Biozym, Oldendorf), detected by ethidium bromide staining and documented with the ‘Easy Plus’ System (Herolab, Wiesloch). The electrophoretic buffer was a TBE-buffer (0.89 M Tris, 0.89 M boric acid, 0.02 M Na,EDTAe2H,O). 2.5. Allelic

ladder

The allelic ladder was composed of alleles which were amplified separately and mixed afterwards. Marker bands (i.e. more intensive bands) within the allelic ladder are derived from PCR-products from persons who are homozygous for a HVR allele. 2.6. Nomenclature

HVEs are designated following the nomenclature of Boerwinkle et al. [6]. 2.7. Statistics

The empirical P-values for the exact Hardy-Weinberg test were estimated according to the Markov chain approach of Guo and Thompson [9]. A dememorization phase of 15 000 initial steps was used before the P-value was estimated in 1000 batches of 10 000 replications. To confirm the differences between the Caucasian populations on the one hand and the American Black and the South Asian population on the other hand, a test for homogeneity was done. 3. Results and discussion

Table 1 shows allele frequencies and genotype distribution in a random population sample from Southwestern Germany (n = 505). The 15 detected alleles were apparent in five homozygous and 42 heterozygous allele combinations. The two most common alleles are HVE 37 and HVE 35 with an allele frequency of 0.374 and 0.244, respectively. Fragment sizes range from 592 bp (HVE 25) to 1012 bp (HVE 53). Size differences of 15 bp could reliably be detected (Fig. 1 and Fig. 2). The distribution of genotypes is in agreement with expected values of the exact Hardy-Weinberg test according to the Markov chain approach of Guo and Thompson [9] (P = 0.1051). The heterozygosity index is 78.4%.

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Table 1 3’-ApoB HVR genotype and allele frequency distribution from a Southwestern German population (n = 505) Allele

25

Frequency 25 -

29 -

29 31 33 35 36 37 39 41 43 45 47 49 51 53

31 3

33 35 1 -l-----__-_8 18 1 18 30

36 -

37 -

39 -

41 -

-1

32 1 202 2 88 16 3 ----I 13 12 2 -2-22---

43 -

45 -

47 -

49 -

51 -

53 -

--5 -

3

6

1

12

9 12 23

1

2

3

33

32

1 4 -6

-

-

-

-

1

-

-

4 2

1 -2

-

2

0.001 0.001 0.082 0.069 0.244 0.004 0.374 0.037 0.010 0.002 0.010 0.062 0.087 0.015 0.002

1

-

A comparison with results from further population studies within other European [lO,ll], an American Black [12] and a South Asian population [13] is shown in Table 2. A test for homogeneity was done within the three Caucasian populations (0.2 < P < 0.3), between the German and the South Asian (P = 0.0005) and between the German and the American Black population (P = 0.005).

Concerning the frequency distribution of common alleles, there are no striking differences between Caucasian populations. All populations show a bimodal distribution scheme with peaks at HVE 37 and HVE 49. Except for the rare HVEs, 30 and 36, alleles have an odd number of repeat units. Unlike the European bimodal distribution scheme the Asian one shows its first peak at HVE 35 (and not at HVE 37). The most frequent alleles are HVE 35, 37

bpM

12

3

4

5

6

7

8

9

10

11

Fig. 1. M, molecular weight marker. Lane 1: allelic ladder composed of HVE 49, 47, 45, 35, 33, and 31. Lanes 2, 5,7, 9, 11: allelic ladders (HVE 49, 47, 45, 39, 37, 35, 33, 31). Lane 3: HVE 49 and 31. Lane 4: HVE 47 and 37. Lane 6: HVE 49 and 33. Lane 8: HVE 47 and 35. Lane 10: 45 and 39.

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Fig. 2. Lane 1: HVE 47 and 36. Lane 2: allelic ladder composed of HVE 53, 51, 49, 47, 45, 41, 39, 37, 35, 33, and 31. Table 2 Comparison of allele frequencies from a German (this study), a Spanish [ll], [13] and an American (Black) population [12] VNTR-alleles

15 17 22 25 26 28 29 30 31 32 33 34 35 36 37 38 39 41 43 4.5 47 49 51 53 55

This study (Germany, n = 505)

Gene (Spain, n = 308)

Chakraborty (France, n = 240)

a French [lo], an Asian

Renges (Asia, n = 107)

Hixson (USA (Black), n = 98)

0.005 0.009 0.001

0.004

0.009

0.082

0.002 0.002 0.086

0.065

0.069

0.062

0.054

0.131 0.005 0.061

0.244 0.004 0.374

0.209 0.006 0.363

0.217

0.037 0.010 0.002 0.010 0.062 0.087 0.015 0.002

0.037 0.016 0.002 0.021 0.083 0.083 0.024 0.002 0.002

0.052 0.023 0.002 0.004 0.071 0.077 0.027

0.001

0.404

0.364 0.005 0.248 0.009 0.042

0.042 0.061 0.009

0.031 0.005 0.005 0.005 0.036 0.020 0.046 0.010 0.097 0.005 0.168 0.005 0.199 0.005 0.092 0.097 0.071 0.046 0.026 0.026 0.005

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and 31. Four alleles which could not be found within Europeans, namely the very short alleles 15 and 17 and HVE 32 and 38, were detected among the South Asian population. Three alleles (HVE 41, 43 and 45) which are present in all the other populations under investigation are not apparent within the South Asian population. The Black population has, in comparison to the Asian (14) and the European (17) populations a great variety of alleles (21) and shows no bimodal distribution scheme. Also striking is that there are 8 HVEs (22, 26, 28, 30, 32, 34, 36 and 38) with an even number of repeat units which make a total of 8.6% (Europeans: two HVEs with even repeat numbers (frequency, 0.4%); South Asians: 3, (1.9%)). Additionally, Blacks have higher frequencies (36%) than Europeans (25%) and South Asians (15%) among the larger alleles (~39 repeats) and among the smaller alleles (533 repeats): Blacks 26%) Asians 22%) Europeans 14%. This is due to the fact that among Asians and Europeans the two alleles with the highest frequency (HVE 35 and 37) account for more than 60% (Europeans: 60.4%; Asians: 61.2%) of all HVR alleles (Blacks: 36.7%). Taken together, it is obvious that the three races (Asians, Blacks and Europeans) possessa different genetic pool for the apoB locus and that the apoB locus is a highly informative marker because of its heterogeneity.

Acknowledgments

We would like to thank Dr James Hixson, Southwest Foundation for Biomedical Research, Department of Genetics, San Antonio, Texas, for providing us with the allele frequency data of the American Black population.

References [l] E.H. Ludwig, B.D. Blackhart, V.R. Pierotti, L. Caiati, C. Fortier, T. Knott, J. Scott, R.W. Mahley, B. Levy-Wilson and B.J. McCarthy, DNA sequence of the human apolipoprotein B gene. DNA, 6 (1987) 363-372. [2] H.K. Das, T. Leff and J.L. Breslow, Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions. J. Biol. Chem., 263 (1988) 11452-11458. [3] W. Xiong, E. Zsigmond, A.M. Gotto, L.W. Reneker and L. Chan, Transgenic mice expressing full-length human apolipoprotein B-100. J. Biol. Gem., 267 (1992) 21412-21420. [4] T.J. Knott, SC. Wallis, R.J. Pease, L.M. Powell and J. Scott, A hypervariable region 3’ to the human apolipoprotein B gene. Nucleic Acids Res., 14 (1986) 9215-9216. [5] L.S. Huang and J.L. Breslow, A unique AT-rich hypervariable minisatellite 3’ to the ApoB gene defines a high information restriction fragment length polymorphism. J. Biol. Chem., 262 (1987) 8952-8955. [6] E. Boerwinkle, W. Xiong, E. Fourest and L. Chan, Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3’ hypervariable region. Proc. Natl. Acad. Sci. USA, 86 (1989) 212-216. [7] E.H. Ludwig, W. Fried1 and B.J. McCarthy, High-resolution analysis of a hypervarible region in the human apolipoprotein B gene. Am. J. Hum. Genet., 45 (1989) 458-464.

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[S] S.A. Miller, D.D. Dykes and H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16 (1988) 1215. [9] SW. Guo and E.A. Thompson, Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics, 48 (1992) 361-372. [lo] R. Chakraborty, M. Fornage, R. Gueguen and E. Boerwinkle, Population genetics of hypervariable loci: analysis of PCR based VNTR polymorphism within a population. In: T. Burke, G. Dolf, A.J. Jeffreys, R. Wolff (eds.), DNA-Fingerprinting: Approaches and Applications, Birkhauser Verlag, Basel, 1991, pp. 127-143. [ll] M. Gene, E. Huguet, C. Sanchez-Garcia, P. Moreno, J. Corbello and J. Mezquita, Study of the 3’-apoB minisatellite performed by PCR in the population of Catalonia. Hum. Hered.. 4.5 (1995) 70-74. [12] J.E. Hixson, P.K. Powers and C.A. McMahan, The human apolipoprotein B 3’ hypervariable region: detection of eight new alleles and comparisons of allele frequencies in blacks and whites. Hum. Genet., 91 (1993) 475-479. [13] H.H. Renges, R. Peacock, A.M. Dunning, P. Talmud and S.E. Humphries, Genetic relationship between the 3’-VNTR and diallelic apolipoprotein B gene polymorphisms: haplotype analysis in individuals of European and South Asian origin. Ann. Hum. Genet., 56 (1992) 11-33.