Forensic Science

Forensic Science International I n t e r n a t i o n a l E L S E V I E R 73 (1995) 15-18 ,,,

Simple detection of the inter-a-trypsin-inhibitor (ITI) polymorphism by isoelectric focusing with

direct immunofixation

Anja Martin*, A. Correns, Petra Otremba, G. Geserick

Institute for Forensic Medicine, Faculty of Medicine (CharitY), Humboldt University, Hannoversche Strafle 6, 10115 Berlin. Germany

Received 6 September 1994; revision received 6 January 1995; accepted 10 January 1995

Abstract

The inter-~-trypsin inhibitor (ITI) polymorphism was analysed in a German population, using polyacrylamide gel isoelectric focusing with subsequent direct immunofixation with monospecific ITI antisera. Gene frequencies of ITI* 1, ITI*2, ITI*3, and ITI*4 were calculated to be 0.6150, 0.3753, 0.0078 and 0.0019, respectively. In our study the allele ITI*4 is described for the first time in a German population.

Keyworda: ITI; Genetic polymorphism; lsoelectric focusing; Direct immunofixation; German population

Zussmmenfassung

Der Inter-a-Trypsin-Inhibitor(ITI)-Polymorphismus wurde in einer deutschen Population mittels isoelektrischer Fokussierung auf Polyacrylamidgelen und anschlieBender direkter Immunfixation mit monospezifischen ITI-Antiseren unter- sucht. Ffir die hiiufigen Allele wurden folgende Genfrequenzen berechnet: ITI*I = 0,6150, ITI*2 = 0,3753; ffir die seltenen Allele: ITI*3 = 0,0078 und ITI*4 = 0,0019. In unserer Untersuchungsreihe wurde alas Allel ITI*4 erstmals in einer deutschen Population gefunden.

Schliisselwtrter: ITI; Genetischer Polymorphismus; lsoelektrische Fokussierung; Direkte Immunfixation; Deutsche Population

* Corresponding author.

0379-0738/95F~9.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01708-Q

16 A. Martin et al./ Forensic Science International 73 (1995) 15-18

1. Introduction

ITI was identified in 1962 as an inhibitor of bovine trypsin with electrophoretic mobility between the ctl- and a2-globulins [1]. ITI belongs to the Kunitz-type pro- tease inhibitors and is synthesised in the liver. Normal plasma concentrations are in the region of 5 mg/dL and it is necessary to perform an immunofixation of immunoblot after IEF.

The genetic polymorphism of ITI was first described by Vogt and Cleve [2] in Ger- man and Tyrolian populations. The phenotypes were detected by agarose gel isoelec- tric focusing of sialidase treated serum samples, followed by immunoblotting and immunofixation with monospecific rabbit ITI antisera. Two common alleles (ITI*I and ITI*2) and one rare allele (ITI*3) were detected at an autosomal locus. In this article we report on an improved procedure for ITI determination.

2. Materials and methods

2.1. Serum samples Untreated sera (stored at -20°C for 2 months to 5 years) of 513 unrelated

individuals were analysed.

2.2. Preparation of gels The polyacrylamide (PAA) gels, sized 250 x 125 × 0.5 ram, consisted of 9.5 ml

solution of glycerine and water (195 ml distilled water, 52 ml glycerine 87%), 5 ml PAA solution (14.55 g Acrylamide, Serva; 0.45 g N'N'-Methylenbisacrylamide, Serva at 100 ml, T 5%, C 3%), and 1.2 ml ampholytes (0.8 ml pH 5-8, 0.2 ml pH 7-9, 0.2 ml pH 3.5-10, Pharmacia/LKB). The solution was degassed for 10 min, then 25/~1 Temed and 0.5 ml ammonium persulfate solution (100 mg ammonium- persulfate in 10 ml distilled water) were added. After polymerisation the gels were kept overnight or up to 3 days at 4°C.

2.3. Isoelectric focusing (lEE} IEF was carried out with a Multiphore II system. Sera were applied on the gel with

5 × 10 mm filter papers (Schleicher & Schiill, No. 604) 1 cm from the anode. Elec- trode solutions were 0.25 M CH3COOH for the anode and 0.25 M NaOH for the cathode. IEF was carried out at 1600 V, 10 W, and 10 mA for a total of 4 h 45 rain (45 min prefocusing time, 1 h with filter papers, 3 h without application of papers). The cooling temperature was 10°C.

2.4. Detection The ITI bands were detected by immunofixation with monospecific ITI antiserum

(Dakopatts, Hamburg). A solution of 0.3 ml antiserum and 0.6 ml bidistilled water was distributed on the surface of the gel. The gel was then incubated in a moist chamber for 30 rain at room temperature, then it was washed in a 0.9% NaCI solu- tion overnight. Staining: Serva blue staining solution, 30 rain at room temperature (staining solution: 1 g Serva blue, 180 rnl ethanol, 420 ml distilled water, 100 ml for- malin). Destaining solution A: 2-6 h, B: overnight (destaining solutions: A: 1250 ml

A. Martin et al,/ Forensic Science International 73 (1995) 15-18

Table 1 Distribution of ITI phenotypes in a random sample from Berlin, Germany

17

Phenotype Observed Expected

n % n % X2-test

1 196 38.22 194.04 37.82 0.0199 2- I 233 45.43 236.78 46.15 0.0603 2 74 14.42 72.23 14.08 0.0432 3-1 5 0.97 4.92 0.96 * 3-2 3 0.58 3.00 0.59 * 3 0 0.00 0.03 0.01 * 4-1 1 0.19 1.23 0.24 * 4-2 I 0.19 0.75 0.15 * 4-3 0 0.00 0.02 0.00 * 4 0 0.00 0.00 0.00 *

Sum 513 100.00 513.00 100.00 0.1236

Gene frequencies: 1 0.61501 2 0.37524 3 0.00780 4 0.00195

Sum 1.00000

*Combined = 0.0002387 0.99 > P > 0.975, df= 3

me thano l , 50 ml fo rmal in , 3750 ml dis t i l led water ; B: 1250 ml m e t h a n o l , 3250 ral dist i l led wate r , 500 ml acet ic acid).

3. Resul ts and discussion

Tab l e 1 shows the resul ts o f I T I typ ing o f 513 un re l a t ed G e r m a n indiv iduals . By

c o m p a r i s o n o f the obse rved and expec ted f requenc ies a g o o d H a r d y - W e i n b e r g equ i -

l ib r ium was seen.

v

1 2 3 4 5 6 7 8 9 10 11 12 1 8 1 4 1 5 ~6 17 18 1 9 2 0 Fig. I. Different ITI phenotypes, anode at the top. ITI 1: lanes 3,12,13, and 16; ITI 2: lanes 2,5,10 and 18; ITI 2-1: lanes 1,6,11,14,15,17,19, and 20; ITI 3-1: lanes 4 and 9; ITI 4-1: lane 7; ITI 4-2: lane 8.

18 A. Martin et al./ Forensic Science International 73 (1995) 15-18

Table 2 ITI gene frequencies from Berlin, Germany, in comparison with other population studies

n ITI*I ITI*2 ITl*3 ITI*4 ITi*5 ITI*6

Africa [5] 126 0.564 0.083 0.337 0.004 - - 0.012 Germany [7] 239 0.600 0.393 0.007 Germany [2] 248 0.575 0.417 0.008 - - - - --

Austria [2] 124 0.577 0 . 4 2 3 . . . .

lran [3l 205 0.612 0.354 0.029 - - 0.005 - - Korea [3] 200 0.532 0.422 0.043 0.003 - - --

Japan [6] 400 0.440 0.526 0.030 0.004 - - - - Basques [8l 303 0.548 0.449 0.003 Germany 513 0.615 0,375 0.008 0.002 -- --

(Berlin)

Fig. 1 illustrates the different ITI phenotypes, detected with direct immunofixa- tion after IEF of untreated sera on PAA gel. The ITI bands were easily demonstrated in the same patterns as described previously in different reports [3-5,6]. In addition, ITI 1-4 and ITI 2-4 were observed, which had not been described in a German pop- ulation before, but in Japanese, Korean and African Negroid populations. This might be due to the fact that the numbers of persons investigated in previous publications were smaller than in our report. There is no indication that the two in- dividuals possessing ITI*4 are not Caucasians. Table 2 shows the gene frequencies of different publications in comparison to our study. The gene frequencies are similar to those from previously reported studies from Germany.

The ITI polymorphism was found to be a useful genetic system in paternity testing [4,6], but this system requires caution in disputed paternity cases. Vogt et al. [4] when applied observed a significant deviation from the Hardy-Weinberg equilibrium in children aged < 18 months. Therefore, the ITI system should be used only for paternity testing in cases involving older children.

References

[1] N. Heimburger and G. Schwick, Fibrin agar electrophoresis 1. Description of the method. Thromb. Diath. Haemorrh., 7 (1962) 432-443.

[2] U. Vogt and H. Cleve, A "new" genetic polymorphism of a human serum protein: inter-a-trypsin- inhibitor, Hum. Genet., 84 (1990) 151-154.

[3l U. Vogt, H. Cleve, D.D. Farhud and H.W. Goedde, The ITI system in South Koreans and Iranians analysed by an improved classification procedure. Hum. Genet., 87 0991) 677-679.

[4] U. Vogt, W. Weise and H. Cleve, The examination of the ITI system in disputed paternities. Int. J. Leg. Med., 104 (1991) 201-204.

[5] U. Vogt, L. Gfirtler and H. Cleve, Inter-a-trypsin inhibitor polymorphism in African blacks. Elec- trophoresis, 13 (1992) 337-338.

[6] I. Yuasa, K. Suenaga, Y. Saneshige, N. Tamaki, K. Ito, and K. Okada, Inter-a-trypsin inhibitor (ITI): a useful genetic system in paternity testing. Evidence for polymorphic occurrence of ITI*3 and existence of a new allele, ITI*4. Int. J. Leg. Med., 104 (1991) 197-199.

[7] C. Luckenbach, J. K6mpf and H. Ritter, Genetic polymorphism of Inter-co-trypsin inhibitor (ITI): formal genetic and linkage analyses. Hum. Genet., 87 (1991) 89-90.

[8] O. Garcia, A. Alonso, A. Aguirre, C. de la Rua and C. Manzano, Genetic polymorphism of Inter-a- trypsin inhibitor (ITI) in the Basque country (Northern Spain). Int. J. Leg. Med., 106 (1993) 129-131.