Humangenetik 29, 2 5 1 2 5 3 (1975) © by Springcr-Verlag 1975

Red Cell Esterase-D Polymorphism in the Population of Tuscany Marino Bargagna, R a n i e r i Domenici*, a n d Agorasti Morali** Institute of Legal Medicine, University of Pisa, Italy Received May 20, 1975

Summary. Esterase-D phenotypes were determined in a population sample of Tuscany, Italy. The observed frequencies of the three alleles are: EsD 1 -- 0.856, EsD 2 = 0.143 and EsD a = 0.001. Studies of the pedigree of the propositus's EsD 3--1 family and mother-child combinations confirm an autosomal co-dominant inheritance.

Introduction H o p k i n s o n et al. (1973) testing f a t t y acid esters of 4-methyl-umbelliferone (4-methyl-7-hydroxy-coumarin), as fluorogenic substrates for the s t u d y of starch gel electrophoretic z y m o g r a m s of h u m a n red cell esterases, discovered the existence of a new estcrase, which t h e y designated esterase-D (EsD). T h e y have shown t h a t EsD, present in m a n y tissues, does n o t act on n a p h t h y l substrates, as the other esterases do, a n d it shows a clear po]ymorphism, with 3 electrophoretic isozyme p a t t e r n s (EsD1, E s D 2 - - 1 a n d EsD2), corresponding to 2 c o d o m i n a n t alleles at a n autosomal gene locus. Bender a n d F r a n k (1974) have reported a new p h e n o t y p i c v a r i a n t ( E s D 3 - - 1 ) , which is t h o u g h t to be a n eterozygotic p a t t e r n d e t e r m i n e d b y the rare allele EsD a. The frequency of E s D alleles has been studied i n different populations. I n this s t u d y we are reporting on p h e n o t y p e a n d gene frequencies in a p o p u l a t i o n sample from T u s c a n y . Materials and Methods The sample included 500 random unrelated individuals. Among them there were 394 blood donors and 106 puerperae. Simultaneous tests were made of the blood of the mothers and of the cord blood of their new born children. The haemolysates were prepared by freezing at --20°C and then by thawing the saline washed red cells. Discontinuous, horizontal, starch-gel thin layer electrophoresis on water cooled plates (according to Parkin and Adams, 1975) was performed for 2 hrs, at 16.5 V/cm, with triscitrate-borate-LiOH buffer system. Bridge buffer: 440 mM boric acid - - 40mlV[ LiOIt, pH 7.2. Gel buffer: 6.75 mM tris - - 1.8 mM citric acid - - 2.2 mM boric acid - - 0.2 mlVILiOH, pit 7.4. Staining solution: 4 mg 4-methyl-umbelliferyl-aeetatein 10 m150 mM sodium acetate. EsD bands were visible within 5 rain under U.V. light (366 nm). * Present address : Institute of Legal Medicine, University of Pavia, Italy. ** Research Fellow of the Ministry of Foreign Affairs of Italy. Present address : Veniselion Hospital, Heraklion, Crete, Greece.

252

M, Bargagna et al. Table 1. EsD phenotype and gene frequencies in the population of Tuscany EsD

Phenotypes observed

EsDI EsD2--1 EsD2 EsD3--1 EsD3--2 EsD3

365 125 9 1

Total

500

Gene frequencies

expected

%

~

%

73 25 1.8 0.2

366.3680 122.4080 10.2245 0.8560 0.1430 0.0005

73.2736 24.4816 2.0449 0.1712 0.0286 0.0001

100

500.00

EsD 1 = 0.856 EsD 2 = 0.143 EsD a = 0.001

100.00

y2 ~ 0.3743 for 2 d/0.90 > P > 0.80

Fig. 1. EsD phenotypes. *Cord-blood samples: note the absence of the fluorescent band in the cathodic region

0

I.

|

II EsOD2-1 (1~ Ill.

[]

EsD3-1

l

D~3-1~

EsD1

Es -

Es(~ D3-1

EsD34

Fig. 2. Pedigree of the propositus's EsD3--1 family. I--2 = dead

Results and Discussion The p h e n o t y p e s in our p o p u l a t i o n sample are given in Table 1. The gene freq u e n c y of EsD t is 0.856. This frequency is somewhat lower t h a n i n other E u r o p e a n samples, reported previously b y the other authors (Hopkinson et al., 1973; Welch a n d Lee, 1974; Bender a n d F r a n k , 1974; B e n k m a n n a n d Goedde, 1974; K i i h n l et al., 1974). A m o n g the 500 individuals of the sample, a rare v a r i a n t E s D 3 - - 1 has been detected. I t s isozyme p a t t e r n corresponds exactly with t h a t observed, at first,

Red Cell Esterase-D Polymorphism in the Population of Tuscany

253

Table 2. Mother/child combinations in EsD polymorphism Mothers

Children EsD1

EsD2

1 EsD2

n

EsDI EsO2--1 EsO2

68 7 --

12 12 --

-5 2

80 24 2

n

75

24

7

106

b y B e n d e r a n d F r a n k (Fig. 1). The s t u d y of the family of the propositus (Fig. 2) is consistent with the suggestion t h a t this eleetrophoretie v a r i a n t is the eterozygote p r o d u c t of the c o m m o n allele EsD 1 a n d the rare one EsD 3. The frequency of E s D a in our sample is 0.001. This finding agrees with the analysis of Harris et al. (1974), a b o u t the incidence of rare alleles of enzymes loci in m a n , d e t e r m i n i n g electrophoretie variants. The mother-child m a t e r i a l provides f u r t h e r evidence of a simple Mendelian c o d o m i n a n t w a y of inheritance. As it has been observed b y H o p k i n s o n et al. (1973), the E s D isozyme p a t t e r n is the same a n d as fully developed in children as i n adults. I n the zymograms of mother-child pairs we have noticed t h a t a eathodieal fluorescent b a n d , i n d e p e n d e n t of E s D p a t t e r n , which appeared in the adults, was a b s e n t in the samples of n e w b o r n children. I t s n a t u r e r e m a i n s to be elucidated.

References Bender, K., Frank, R.: Estcrase D-Polymorphismus: Darstellung in der Hochspannungselektrophorese und Mitteilung yon Allelhiiufigkeiten. Humangenetik 23, 315--318 (1974) Benkmann, H.-G., Goedde, H. W. : Esterase D polymorphism: Gene frequencies and family data. Humangenetik 24, 325--327 (1974) Harris, H., Hopkinson, D. A., Robson, E. B.: The incidence of rare alleles determining electrophoretic variants: data on 43 enzyme loci in man. Ann. hum. Genet. 37, 237--253 (1974) Hopkinson, D. A., Mestriner, M. A., Cortner, J., Harris, H. : Esterase D : a new human polymorphism. Ann. hum. Genet. 37, 119--137 (1973) Kiihnl, P., Nowicki, L., Spielmann, W. : Untersuchungen zum Polymorphismus der intraerythrocyti~ren Esterase D (EsD) mittels Hochspannungselektroforese auf Agarosegel. Z. Rechtsmedizin 7~, 179--182 (1974) Parkin, B., Adams, E. : Med. Sci. Law (1975, in press) Tashian, R. E. : The esterases and carbonic anhydrases in human erythrocytes. In: Biochemical methods in red cell genetics (ed. J. J. ¥unis), pp. 307--336. New York-London: Academic Press 1969 Welch, S. : Red cell esterase I) polymorphism in Gambia. tIumangenetik 21, 365 367 (1974) Welch, S., Lee, J.: The population distribution of genetic variants of human esterase D. Humangenetik 24, 329 331 (1974) Prof. Marino Bargagna Istituto di Medicina Legale Via Roma, 55 1-56100 Pisa, Italy