α-l -Fucosidase phenotyping in human placentae, semen and seminal stains

37

Forensic Science International, 30 (1986) 3743 Elsevier Scientific Publishers Ireland Ltd.

(u-L-FUCOSIDASE PHENOTYPING AND SEMINAL STAINS

IN HUMAN PLACENTAE,

AKIRA KIDO, NOR1 KOMATSU and MASAKAZU

SEMEN

OYA

Department of Legal Medicine, Yamanashi Medical University, Yamanashi-ken 409-38 (Japan)

Tamaho-machi,

(Received July 12,1985) (Accepted October 17,1985)

Summary The polymorphism of a-L-fucosidase (Fu) was investigated in a Japanese population from samples of placentae and semen, using isoelectric focusing. The gene frequencies of placental types were Fu’ = 0.748 and Fu’ = 0.252, and those of seminal types were Fu’ = 0.739 and Fu’ = 0.261. The coincidence in the distribution between the placental and seminal types suggests that the Fu types occurring in placentae and in semen are controlled by the same Fu alleles. The Fu typing was possible in seminal stains stored at 4’C for up to 9 weeks, at room temperature for up to 7 weeks and at 37°C for up to 4 weeks. The Fu types were still detectable at semen dilutions of up to 1 :4. This polymorphism would provide a useful genetic marker for the medicolegal grouping of seminal stains. Key words: stains

a-L-Fucosidase;

Polymorphism;

Japanese population;

Phenotyping in seminal

Introduction The genetic polymorphism of human cu-L-fucosidase (Fu) was first discovered in leucocytes by Turner et al. [ 11. Using isoelectric focusing in polyacrylamide gels, they demonstrated three phenotypes (Fu 1,2-l and 2), which are determined by a pair of codominant autosomal alleles Fu’ and Fu’. Subsequent studies showed that this polymorphism occurs in most tissues and urine [l-3]. Recently, Gill and Sutton [4] reported the occurrence of the Fu types also in human semen and other body fluids. In the present study the distribution of Fu types in the Japanese was examined utilizing the placenta and semen as the source of the enzyme. Furthermore, the phenotyping of Fu in seminal stains was investigated for medicolegal use. Materials and methods Platen tae Human full term placentae from normal single births of 226 Japanese 0379-0738/86/$03.50

0 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

38

parturients were provided by the Department of Obstetrics and Gynecology, Yamanashi Prefectural Central Hospital and stored at -20°C. A small piece of placental tissue was minced and homogenized in an equal volume of distilled water using an Ultra-Turrax homogenizer (Junke & Kunkel AG, Staufen in Breisgau, F.R.G.). The homogenates were centrifuged at 3000 rev./min for 10 min and the supernatants were retained for analysis. Semen Ejaculates were collected from 157 male patients at the Fertility Clinic of Yamanashi Prefectural Central Hospital. They consisted of 52 samples of normospermia (sperm counts over 40 X 106/ml), 69 samples of oligospermia (sperm counts under 40 X 106/ml) and 36 samples of azoospermia. The samples were stored at -20°C until use. Neuraminidase treatment Ten microliters of 500 U/ml neuraminidase (BDH, England) and 2.5 ~1 of 10 mM citratephosphate buffer (pH 4.8) were added to 10 ~1 of the sample. After the incubation for 17 h at room temperature the mixture was subjected to electrophoresis. Seminal stains Seminal stains (10 samples of normospermia and 10 samples of oligospermia) were made on filter paper (Toyoroshi No. 2, Tokyo, Japan). They were stored in a refrigerator at 4”C, at room temperature and in a thermostatic chamber at 37”C, and examined weekly over a period of 15 weeks. Isoelectric focusing Isoelectric focusing was performed essentially by the method of Gill and Sutton [4] with an ATT0 SJ-1071 isoelectric focusing apparatus (Tokyo, Japan). The gel plate (200 X 150 X 0.5 mm) was composed of 20 ml of stock solution (5.25% acrylamide/0.25% N,N’-methylenebisacrylamide), 1 ml of Ampholine pH range 5-7 (LKB), 0.3 ml of 0.01% riboflavine and 2.5 g sucrose. The electrode paper strips were soaked with 1 M phosphoric acid for the anode and with 1 M sodium hydroxide for the cathode. Placental extracts and semen samples were applied to the gel surface 1 cm from the anode using 5 X 6 mm filter paper. Seminal stains were cut in 5 X 6 mm pieces, moistened with a minimal amount of distilled water and directly applied to the gel plate. Electrofocusing was conducted at a constant voltage of 2000 V for 180 mm. During focusing the gel plate was cooled by circulating water at 4°C. Staining After electrofocusing the gel was stained by the method of Turner et al. [5]. A piece of filter paper strip (Whatman No. 3, U.K.) was soaked in

39

0.1 M citrate-phosphate buffer (pH 4.8) containing 0.2 mg/ml of 4-methylumbelliferyl-cu-L-fucoside (Nakarai Chemicals, Kyoto, Japan) and applied onto the entire surface of the gel plate. The gel plate was wrapped in a sheet of thin plastic film and incubated at 37°C for 60 min. Then, the gel was placed in a chamber containing ammonia gas for a few minutes. The Fu isoenzymes were visualized under long-wave ultraviolet light. Results and discussion Electrophoretic pattern Figure 1 shows the isoelectric focusing patterns of Fu types in placentae and semen. In placentae the three common phenotypes were identified without difficulty. The Fu 1 type exhibited two major bands while the Fu 2 type three major bands. The Fu 2-1 type consisted of five major bands: two from the Fu 1 type and three from the Fu 2 type. Each phenotype had additional minor bands towards the anode.

(+I r---,r--7

II

I

r__3r--Tr--7

I

L__JL-__I

L__JL--JL--J r---r---,

r--7

r--3L-_J L~~J~~~~~~~~

L__.lL__J

r--i

?---7r--7l___J

r--7

L__JL--JL--JL_-JL__d

mm

mm

I::

,__I

r---l I--

---f

l._

__J

L__~

r--7

L-_-A

1

2-l

Placentae

2

1

2-1

Semen

2

1

2-l

PlCacentae

2

1

2-l

SemE !n

Fig. 1. Isoelectric focusing patterns of Fu tyr be6in placentae and semen.

2

40

The electrophoretic patterns of Fu types observed in semen closely resembled those of the corresponding types in placentae. However, the homozygous Fu 1 and Fu 2 types in semen exhibited two minor bands towards the cathode. These bands were not visible in the heterozygous Fu 2-l type. Our observation is in the main consistent with that of Gill and Sutton [4]. There were no differences in electrophoretic pattern among normospermia, oligospermia and azoospermia. According to the suggestion of Turner et al. [l] that Fu types in leucocytes and tissues can be easily distinguished by treatment with neuraminidase, we examined and compared its effects on the isoenzyme patterns. Neuraminidase treatment of placental extracts as well as semen caused a reduction in staining intensity of the anodal minor bands, which supports the view of Turner et al. [l] that these bands result from the attachment of sialic acid residues to the primary enzyme form. However, reliable typing was possible without the aid of neuraminidase treatment. Distribution

of Fu types

The distribution of Fu types examined in 226 placentae from Japanese parturients is shown in Table 1. No rare variants were observed in our samples. The observed numbers did not deviate from the expectation according to the Hardy-Weinberg law. Table 2 shows the distribution of Fu types observed in 157 semen samples from Japanese males, which is practically the same as that in placentae. The complete coincidence in the distribution between the placental and seminal types strongly suggests that the Fu types occurring in placentae and in semen are controlled by the same Fu alleles. Our results are almost similar to those for another Japanese subpopulation examined in leucocytes by Toyomasu et al. [6]. The Fu’ gene frequency for the present Japanese population (0.252) is not significantly different from that for Europeans [7-lo], except Poles (0.347) [ll] and French (0.36) [12], but it is much higher than that for Black Americans (0.074)

PI. TABLE

1

DISTRIBUTION

OF PLACENTAL

Phenotype

No. observed

1 2-1 2

131 76 19

Total

226

Fu TYPES IN JAPANESE

Fu’ = 0.748.

Fu2 = 0.252. x2 = 2.629,

d.f. = 1,0.20

> P > 0.10.

f%) (58.0) (33.6) (8.4) (100.0)

No. expected 126.4 85.2 14.4 226.0

41 TABLE

2

DISTRIBUTION

Phenotype

OF SEMINAL

No. obserued

Fu TYPES IN JAPANESE

No. expected

(%) Total

Normospermia

Oligospermia

Azoospermia

1 2-l 2

24 21 7

41 27 1

22 10 4

87 (55.4) 58 (36.9) 12 (7.6)

85.7 60.6 10.7

Total

52

69

36

157 (99.9)

157.0

Fu’ = 0.739. Fu’ = 0.261. xz = 0.290,

d.f. = 1,0.70

> P > 0.50.

Determination of Fu types in seminal stains Table 3 summarizes the results for the determination limits of Fu types in 20 seminal stains stored at 4”C, room temperature and 37°C. All the seminal stains examined could be typed for Fu at 4°C for periods of up to 9 weeks, at room temperature for periods of up to 7 weeks and at 37°C for periods of up to 4 weeks, although the isoenzyme patterns observed in seminal stains were in general not so clear as compared with those in seminal fluid. The bands became fainter and more indistinct with increasing time TABLE

3

POSITIVE RESULTS FOR THE DETERMINATION PHENOTYPES IN 20 SEMINAL STAINS STORED AND 37°C

Temperature

Phenotype

LIMITS OF Fu 1, 2-1 AND 2 AT 4”C, ROOM TEMPERATURE

Age of seminal stains (weeks) 1

2

3

4

5

6

7

8

9

10 II

12 13 14 15

4°C

1 2-l 2

12 12 12 12 12 666666666444333 222222222222222

12

12

12

12

12

11

11

10

10

10

Room temperature

1 2-1 2

12 12 12 12 12 666666664433111 22222222221110

12

12

11

10

10

9

9

8

7

7

37°C

1 2-1 2

12 12 12 12 11 666654310 2222222222110

11

11

10

7

6

5

5

4

3

3

Fig. 2. Isoelectric focusing patterns of Fu types in seminal stains stored at room temperature for 1 week (A) and for 4 weeks (B). of storage, particularly in the heterozygous phenotype Fu 2-l. The isoelectric focusing patterns of Fu types in seminal stains stored at room temperature for 1 week and for 4 weeks are shown in Fig. 2. The present stability study shows that the Fu typing is possible from seminal stains TABLE 4 POSITIVE RESULTS FOR THE DETERMINATION LIMITS OF Fu 1, 2-l AND 2 PHENOTYPES IN FRESH STAINS FROM 30 SEMEN SAMPLES SERIALLY DILUTED WITH PHYSIOLOGICAL SALINE Phenotype

No. tested

Dilution I:2

1:4

1:8

1:16

6 2 0

0 0

--1 2-1 2

14 12 4

14 12 4

12 11 4

43

after fairly long storage periods. This polymorphism would provide a useful genetic marker for the medicolegal grouping of seminal stains. However, owing to the appearance of the minor bands control samples from leucocytes or placental extracts are required in trying to use this polymorphic system in any forensic casework. Table 4 shows the results for the determination limits of Fu types in fresh stains from 30 semen samples serially diluted with physiological saline. Most of the samples examined were correctly typed for Fu at dilutions of up to 1: 4. The typing was no longer successful at a dilution of 1: 16. The present results of dilution experiments are of practical value in the examination of vaginal swabs in rape cases, since ejaculates are to some extent diluted with secretions within the vagina. However, it should be taken into account that the Fu isoenzymes can be detected also in vaginal fluid [4]. Acknowledgement We are very grateful to Dr. M. Saito, Yamanashi Prefectural Central Hospital, for supporting this work. References 1 B.M. Turner, V.S. Turner, N.G. Beratis and K. Hirschhorn, Polymorphism of human OLfucosidase. Am. J. Hum. Genet., 27 (1975) 651-661. 2 E.M. Beyer and G.Y. Wiederschain, Further evidence of human a-L-fucosidase polymorphism. Clin. Chim. Acta, 123 (1982) 251-259. 3 K. Kishi and T. Yasuda, a-L-Fucosidase polymorphism in human urine revealed by isoelectric focusing. Proc. Jpn. Acad., 61 (1985) 186-189. 4 P. Gill and J.G. Sutton, or-L-Fucosidase polymorphism in human semen, blood, and vaginal fluid. Hum. Hered., 34 (1984) 231-239. 5 B.M. Turner, N.G. Beratis, V.S. Turner and K. Hirschhorn, Isozymes of human a-L-fucosidase detectable by starch gel electrophoresis. Clin. Chim. Acta, 57 (1974) 29-35. 6 T. Toyomasu, K. Suzuki and H. Matsumoto, Genetic polymorphism of oc-fucosidase in a Japanese population. Act. Crim. Japon., 50 (1984) 11-12. 7 G. Corney, R.A. Fisher, P.J.L. Cook, J. Noades and E.B. Robson, Linkage between cx-fucosidase and the rhesus blood group. Ann. Hum. Genet., 40 (1977) 403-405. 8 I’: KuhnI and W. Spielmann, Neuere EntwickIung der Elektrofokussierungstechnik. Arztl. Lab., 27 (1981) 255-260. 9 J. Henke and T. Netzer, Forensic application and polymorphism of the human lysosomal or-fucosidase (EC 3.2.1.51). Forensic Sci. Znt., 19 (1982) 107-111. 10 M. Giannetti, Untersuchung zum Polymorphismus der Alpha-L-Fucosidase (EC 3.2.1.51) im Raum Kiiln-Bonn. Anti. Lob., 29 (1983) 300-302. 11 Z. PrzybyIski, T. Dobosz and M. Stawarz, Genetic polymorphism of FUC (EC 3.2.1.51) in Polish population. 2. Rechtsmed., 89 (1982) 21-24. 12 I. Trinh-Dinh-Khoi, D. Glaise, A. Le Treut, R. Fauchet, Y. Godin and J.Y. Le Gall, Genetic polymorphism of a-L-fucosidase in Brittany (France). Hum. Genet., 51 (1979) 293-296.