Ann. Hum. Genet., Lond. (1964), 28, 39 Printed in Great Britain

39

Effects of locus MN on diastolic blood pressure in a human population

BY R. CRUZ-COKE,* R. NAGELt AND R. ETCHEVERRYS School of Medicine of the Univeraity of Chile

Many authors have shown that arterial blood pressure is inherited as a graded character (Pickering, 1962; Miall & Oldham, 1957; hz-Coke, 1960a, b ; 1963 andearlier writers; Stocks & Karn, 1924). This does not, however, exclude the possibility that a single pair of allelomorphic genes may influence this metrical trait. Intelligence also seems to be inherited polygenically, and yet it is known that some individuals with the lowest intelligence pin a population sample may occupy the extreme tail of the distribution curve by virtue of a single gene defect, e.g. phenylketonuria. We cannot say to what extent the side effects of major or polymorphic genes play their part in determining multifactorial variation. Their effects in metrical traits are almost unexplored and their importance as a source of variation is unknown (Falconer, 1960).

It is not easy to apply to man the methods of quantitative genetics. As Fraser Roberts (1961) states, we are limited to little more than measures of resemblance between close relatives. The analysis of theoretical measures of resemblance is possible, but the conditions under which theoretical values are realized are difficult to fulfil, because environmental factors influence the degrees of resemblance to an appreciable extent. As Li (1961) pointed out, in quantitative genetics, a satisfactory method of analysis of the interaction between genotype and environ- mental components has yet to be developed.

The genetics of arterial blood pressure centres round the study of its phenotypic variation (Falconer, 1960). As we know, the amount of variation is expressed as variance. Now, roughly, the phenotypic variance is the sum of genotypic and non-genotypic variance, when all the genotpes react the same way to different environmental conditions. Unfortunately, these conditions do not occur in nature. In order to overcome this difficulty i t is necessary to choose as far as possible a true random sample of an isolate population living under the most uniform environment (Li, 1961), and also to observe the type of reaction of a given genotype under the influence of different powerful environments.

Assuming that the basis of our work is monofactorial it is possible to study the effect of a single locus on blood pressure. It seems that the MN system would be useful for this purpose, because its genotypes are simply determined and the gene frequencies of both alleles are more or less equal and it is very stable in its world-wide distribution (Reed, 1962). This fact is very important because genes contribute much more variance when at intermediate frequencies (Falconer, 1960).

The purpose of this investigation is to analyse the possibility that some major genes, denoted by L M and LN, may be a source of variation of blood pressure in a human population.

* Division of Medicine, Hospital Jose Joaquin Aguirre and Department of Genetics, Institute of Biology. t Institute of Physiology. Present address : Department of Medicine, Albert Einstein College of Medicine,

$ Department of Hematology, Hospital del Salvador. Yeshiva University, New York.

40 R. CRUZ-COKE, R. NAGEL AND R. ETCHEVERRY

THE DATA AND METHODS

During 1963 an expedition of the School of Medicine of the University of Chile undertook a medical and anthropological survey of the native population of Easter Island (109” 27‘ W.; 27’ 09’ S.) (Cruz-Coke, 1963; Cruz-Coke & Iglesias, 1964; Cruz-Coke, Etcheverry & Nagel, 1964; Nagel, Etcheverry & Guzman, 1964). From the 1079 natives who inhabited that island in January 1963, a random sample of 179 subjects was collected and examined there during the survey. The environmental and genetical conditions of the sample were the following.

(a) Environmental conditions The population of Easter Island is influenced environmentally by a process of migration.

Every year a stream of natives emigrates to the mainland, and other natives return to the island. From the point of view of migration, there exist two types of natives : (a) the ‘islanders ’ who remain permanently. in their ecological habitat, and (a) the ‘continentals’, who migrate. Consequently the two groups have lived under two definitely different environments (Cruz- Coke et al. 1964). It was possible to observe this environmental difference when reading blood pressure in the two migration groups. A single observer took measurements of blood pressure on a random sample of 10 years and older, according to techniques reported elsewhere (Cruz- Coke & Covarmbias, 1964). The sample was divided into 129 ‘islanders’ and 50 ‘continentals’. A regression analysis of diastolic blood pressure on age was worked out in each group according to the method proposed by Dixon & Massey (1957). Only diastolic figures were used for reasons given (Cruz-Coke, 1 9 6 0 ~ ) . Table 1 shows that the two regression coefficients are different. The ‘continental’ blood pressure variable Y is highly dependent on the age variable X. On the contrary, the ‘islander’ variable Y is independent of the X variable. This analysis shows that the migration process influences significantly the dependence of blood pressure on age of the Easter islanders. Our sample has been clearly submitted to two different powerful environ- ments.

Table 1. Regression analysis of diastolic blood pressure ( Y ) on age ( X ) ‘ Islanders ’

Number of subjects 129 Mean age, years (X) 32.8 Standard deviation 17-08 Mean diastolic blood pressure in mm Hg ( Y ) 84.2 Standard deviation 9’42 Regression coefficient, b + 0.09 Test for linearity of regression 1.41

( P > 0.05 for 4, 123 D.F.)

I -87 ( P > 0.05)

Test for independence of Y on X

( b ) Genetical analysis

‘ Continentals ’

5 0 344 I444 86.8 12.15

+ 0’34 0.24

( P > 0.05 for

3.08 4, 4.4 D.F.)

( P > o*oor)

The genetical structure of the total sample was studied through the following polymorphic systems: (1) Alleles from the blood groups ABO, MN, Rh, Kell, Diego, which were classified within 12 hr. in the laboratory situated in the island. (2) Alleles Hp’ and Hp2 for phenotypes of haptoglobins, which were determined by starch gel electrophoresis according to the technique of Smithies (1955) and Nagel & Etcheverry (1963). The sera were separated the same day and stored at -20’ C. until they were examined and classified. (3) Alleles T, t from P.T.C. taste

Efects of locus M N on diastolic blood pressure in a humn population 41 sensitivity according to the sorting test of Harris & Kalmus (1949) using fifteen solutions. (4) Colour blindness was determined with the test of Ishihara (1954) and the pseudoisochromatic plates of Hardy, Rand & Ritter (1957). Gene frequencies of blood group alleles were deter- mined according to the methods proposed by Race & Sanger (1962). The gene frequencies of the MN and haptoglobin systems were determined according to the methods of Cotterman for related samples (Nagel & Etcheverry, 1963).

Table 2. Determination of the gene frequency of the allele LM in two groups, according to the method of Cotterman (Nagel & Etcheverry, 1963)

n MM MN N N Total x Y W wx WY a b c za+b z ( a + b + c ) 2/(92+1)

Islanders Unrelated - 12 19 18 49 43 98 1.0000 43 98 No. related 2 3 18 13 34 24 68 0.6667 18 45.2

No. related 4 I 2 I 4 4 8 04000 1-6 3-2

Totals 113 71’9 170’7

No. related 3 4 10 7 21 I8 42 0.5000 g 21

No. related 5 o ‘ 4 5 I I 0 0.3333 0.3 3’3

71’9 170.7

LM ( p ) = - = 0.42 0.031 x2 = 0’947 ( P > 0.10)

Continentals Unrelated - 8 8 7 2 3 24 46 I*OOOO 24 46 No. related 2 3 11 2 16 I7 32 06667 11.3 21.3 No. related 3 I 5 0 6 7 12 0.5000 3.5 6.0

Totals 45 38.8 73’3 38-8 73’3

L M ( p ) = - = 0.53 f 0.054 x2 = 0.225 (P > 0.10)

A preliminary analysis of consanguinity in the entire population of the island was performed by the method of Wright (Stern, 1960) according to data collected there (Cruz-Coke & Iglesias, 1964).

( c ) Analysis of variance The effect of locus MN on diastolic blood pressure was investigated by analysis of variance

according to the methods proposed by Falconer (1960) and Li (1955). The environmental effects of variance were calculated according to Li (1961).

RESULTS

(a) Nature of the sample Table 2 shows the determination of the gene frequency of the allele L M , according to the

method of Cotterman. Neither of the gene frequencies differ significantly and their genotypes were in equilibrium conditions according to Fisher’s test. Table 3 shows in both migration groups similar gene frequencies for all blood group alleles. All the respective genotypes were in equilibrium conditions. This study was made on only 113 ‘islanders’ and 44 ‘continentals’. In the group of 50 continentals who were tested, we found only five ‘non-tasters’ for P.T.c.,

and in the ‘islander’ group, seven ‘non-tasters’ (solution < 4). This difference is not significant (2’ = 1.20, P > 0.05).

42 R. CRUZ-COKE, R. NAGEL AND R. ETCHEVERRY

Table 3. Gene frequencies for alleles of the ABO, M N , Rh, Kk, Di and H p locus in two

Allele

I* I= I? L' cde(r) K Dib HP'

groups of the sample of Easter Islanders Islanders Continentals

f A h 7 f I

n Gene frequency x* n Gene frequency x2 46 46 46 44 44 44 44 45

P

> 0'10 > 0'10 > 0'10 > 0'10 - - -

> 0'10

x2, genic equilibrium for genotype distribution. P, level of significance of differences.

Table 4. Distribution of the tribal ancestry of two groups of Easter Islanders Islanders Continentals

A A I \ f >

he-existing tribe n Yo n % Miru I7 13.1 7 15.3 Marama 29 22.6 8 17.3

Ngaure 30 23.6 9 19.5 R&a 7 5 '4 5 Ngatimo 2 I ' 5 0

Non-tribal I 2 9 3 2 4'3 Totals 129 100'0 46 100'0

Tupahotu 32 24'5 15 32.8

10.8 -

Table 5 . Nean dimtolic blood pressure phenotyph values of the locus M N in two groups of Easter Islan.ders

Continentals Islanders .A

T A

7 f \

Diastolic blood pressure mm Hg

Mean S.D.

Diastolic blood pressure mm Hg - -

Phenotypes No. Mean S.D. No. MM 20 81-7f 6.90 I 2 77'0f 7.82* MN 50 846 f I 1-22 24 89.7 k I 1-62 NN 43 85'3f 8'33 9 93'7 f 13.63

Totals 113 45

Significant difference P > 0-001 (Welch procedure).

Colour blindness waa found only in two malea who were brothers and children of a Tahitian immigrant. Both were deutan. The sixty-five males of the 'islander' groups showed two colour blind, and not one was found in the thirty 'continental' males. This difference is not significant (x2 = 0.26, P > 0.10).

Only one (0.62%) of the 160 marriages of the total native population of Easter Island was found to be a consanguineous union (second cousin once removed). This couple had three children.

Effects of locus M N on diastolic blood pressure in a human population 43 It is possible to explain this very low degree of consanguinity, considering the existence of

Table 4 shows a very similar distribution of tribal ancestry in both migration groups. ‘taboo ’ in this population, which forbids consanguineous unions (Mhtraux, 1940).

50

40

30

20 10

0

x

60 i. - - - - -

60

50

40-

30

20

10

s

mm Hg

Continental sample - -

- - -

)kO

30 4 50 60 70 80 90 100 110 120 130 mm Hg

Fig. 1. Frequency distribution curves of diastolic blood pressure for phenotypes of locus MN; MM, interrupted line; MN, continuous line; NN, dotted line. Males and females.

(b ) Analysis of variance

Table 5 shows that diastolic blood pressure values for phenotypes MM, MN and NN are different in the two groups. In the ‘islander’ group the three mean values do not differ signifi- cantly; the three genotypes are confounded. On the contrary, in the ‘continental’ group homozygotes MM and NN are separated at a high level of significance (P > 0.001 with 1 6 1 5 degrees of freedom), according to the Welch procedure for ‘Two sample t-test’ for unequal variances (Brownlee, 1960). Fig. 1 shows that the two homozygotes MM and NN are shifted in opposite directions to the ends of the tails of the curve under the effect of a new environment.

Table 6 shows the analysis of variance and linear regression for locus MN and diastolic blood pressure. In the two migration groups, the dominance deviation is small and the parent-child genetic correlation coefficient reaches a significant value of 0-44 and 0.46. Fig. 2 shows in both groups the regression of diastolic blood pressure genotypic values ( Y ) on genotypes for locus MN (X). In Fig. 2 B, in spite of the shifting of the two homozygotes to higher and lower values, the linear or additive component of the genotypic variance maintains an arithmetic pro- gression and shows a small dominance deviation. Consequently, the genetic correlation coefi- cient 0.44 remains nearly unchanged (0.46).

Table 7 shows in both groups a rough partitioning of the total vmiance. Random environ- mental influences increased the phenotypic variance to a$ = a: + dE, and hence the correlation

44 R. CRUZ-COKE, R. NAOEL AND R. ETCHEVERRY waa decreaeed to +(ai/bsp). Consequently the phenotypic correlation coefficient was diminished to very low values. Nevertheless, hereditability (&a;) which is the proportion of the total variance of blood pressure which is attributable to the effects of a pair of genes LM and UV, reached, in the ‘continental’ group, a significant value (0.24).

0 1 2

X (MM) ( M N (“1

Fig. 2. Regression of mean blood pressure phenotypic values (Y) on genotypes for locus MN ( X ) . Black circles, actual values. White circles, fitted values. 0, mean. Males and females.

DISUUSSION

We have obtained a sample with a random geographical distribution, and individuals of all the twenty-four families of the six pre-existing tribes were represented there (Cruz-Coke & Iglesias, 1964). The genotypic proportions of the alleles investigated were distributed in equili- brium conditions. When the sample was divided according to the process of migration, the gene frequencies for all the allelee studied remained unchanged in both groups. We think, consequently, that our sample is roughly genetically comparable, and useful as a study of a population under the influence of two different powerful environments.

We have shown that the blood pressure variance of a population is influenced directly by the duration and intensity of the environmental factors and is irrespective of age (Cnrz-Coke, 1960b). Now, in this sample, we have shown that the change of environment increased the variance of blood pressure of Easter Islanders when they reached the continent (Cnrz-Coke et al. 1964). But we were interested also in hd ing out what change there would be in the distri- bution of phenotypes MM, MN and NN of a similar population on increasing the variance from 92.4 to 133.8 mm Hg. Our results showed that in the ‘islanders’, the phenotypes were con- founded, but in the ‘continentals’, with an increased variance, the two homozygotes had reacted in different and opposite ways. The tendency of the homozygote NN to become ‘hyper- tensive’ in a new environment is very strong; all the individuals who became ‘hypertensives’ on reaching the continent carried the allele LN in its genotype. Fig. 1 shows clearly that homozygote MM tends to be ‘hypotensive’ and the heterozygote to remain at intermediate range.

Eflects of locus M N on diastolic blood pressure in a human population

Table 6. Analysis of genetic variance of blood pressure and locus M N into an additive genic m p o n e n t and a component due to dominance deviation

45

units of genic Actual Fitted

Frequency effect value value Genotype f X Y fw L

(Origin 70 md3.g) Islanders " 0'34 2 15-34 '0.43 15-75 M N 0.48 I 1460 7-00 14.07 M M 0.18 0 11-75 0 12-39 SUm I '00

1-16 I433 16-62 14.33 Mean - Covariance - 0.48 1.51 0.81 1'35

- 17-43 - -

Continentals N N 0.23 2 23'75 10.92 25'96 M N 0 5 0 I I970 9-85 17'74 M M 0.27 0 7-70 0 9.62 sum I '00

Mean L 0.96 17'41 I 6-7 I 17.41 Covariance - 0.50 35'43 406 32-96

- 20.77 - -

Genetic correlation coefficient: islanders, r 0.44; continentals, r 0.46.

Dominant component

D

+0.41 - 0'53 + 0.64 -

0

0.16

+ 2'21 - 1-96 + 1.92 - 0

2'47

Table 7. Partitioning of the phenotypic variance of blood pressure into m p o n e n t s attributable to different cawm

Islandem Continentals

Number of subjects Gene frequency

Allele uu Allele L M

Average gene effect (a) Partitioning of vmianoe

Additive component Dominant component

113 45

0.58 0.48 0.42 0'52 I a68 8-24

1-35 32-96 0.16 2'47

Genotypic variance 1.51 35'43 Environmental variasloe 90.90 98.22 Phenotypic variance 92.41 133.65

Parent-child correlation Genetic Phenotypic Hereditability

0447 0.465 0.007 0.123 0.014 0.246

We cannot determine the nature of this process of interwtion between genotypes and en- vironment, The analysis of variance shows that the genotypes of the locus MN in a new genera- tion of Easter Islanders are created afresh with genes L M and UV transmitted by an older generation, mainly through an additive process, because, in both groups the genetic correlation coefficient reaches a significant value of 0.44 and 0.46 (expected value 0.60). Nevertheless, we cannot make the assumption that any of the LM and LN genes acts additively, in spite of the almost complete absence of dominance (Falconer, 1960).

46 R. CRUZ-COKE, R. NAGEL AND R. ETCHEVERRY We have worked with a single locus. We could not study the interaction deviation with

another locus, as the Hp system, for two reasons; first, in Easter Island the gene frequency of the allele Hpl appears to be the highest in the world, reaching a value of 0432. So, gene Hp2 at a very low frequency has a limited contribution to variance. Secondly, in Easter Island we have found only one phenotype Hp2-Hp2 and we could not score blood pressure in the same way as with the MN system. Consequently, it was not possible to make the joint analysis of variance with locus MN and Hp. We could only demonstrate that there exists a very small within-locus interaction (dominance deviation) in the MN system and blood pressure.

We must consider many complications in connexion with the partitioning of the variance (Falconer, 1960). First, the inbreeding of the population influences the environmental variance. Fortunately in the population of Easter Island the degree of consanguinity is very small. Another important complication is the existence of a probable correlation between phenotypic value and environmental deviation, which introduces a genotype-environmental correlation. In our sample it seems to us unavoidable and it is difficult to anslyse this problem. Consequently we considered any covariance term that may arise from genotype-environmental correlation as being part of the genotypic variance. A third important complication is the problem of the genotypic-environmental interaction. In our sample a specific difference of environment bas a greater effect on some genotypes than on others; for instance, the ‘continental’ environment increases the number of blood pressure phenotype NN and lowers that of MM. Undoubtedly, this is a process of interaction between genotype and environment, and gives rise to an addi- tional component of variance. Since it cannot be meaeured separately, it is best regarded as part of the environmental variance (Falconer, 1960). So, in the cme of the variance partitioning of the ‘continental ’ group, the environmental component, which reaches the value of 75 % of the total variance, could be divided roughly into two parts : an environmental component and the genotype-environmental interaction.

Having considered these complications we must interpret Table 7 very carefully. Obviously, the influence of environment has decreased the correlation coefficients to very low values. Nevertheless, in the ‘continental’ group, roughly the 25 yo of the phenotypic or total variance could be attributed to the additive component of the genetic variance. The change of environ- ment operating on the same set of genotypes had shown that there is a hidden effect of a polymorphic gene in the process of the blood pressure genetic-environmental interaction. In spite of the limited conditions of our investigation, it seems to us that locus MN is a ‘relatively important ’ source of variation of the blood pressure phenotype in the population of Easter Island.

In a general population, some of the major genes, as polymorphic ones, may have pleiotropic or secondary effects on metric characters. Our investigation seems to support the possibility that polymorphic genes may contribute at a relatively important level to the variance of blood pressure in a human population. The alleles of some of these loci are at intermediate frequencies, and they are probably maintained by selection favouring heterozygotes, with a fairly small effect on fitness. Unfortunately, it is difficult to probe this suggestion because we can only study this problem by indirect means.

SUMMARY

1. In an analysis of variance for locus MN and arterial diastolic blood pressure in a human population, we have studied a random sample of 179 inhabitants of Easter Island, existing under the influence of two different powerful environments.

Effects of locus M N on diastolic blood pressure in a human population 47

2 . The process of migration to the mainland influences significantly in this population the regression of blood pressure on age, increasing its variance.

3. The mean blood pressure phenotypes for MM, MN and NN are confounded in the popu- lation which stayed in the island. On the contrary phenotypes MM and NN of the emigrated population were found to be significantly different from one another.

4. The respective analysis of covariance and linear regression shows a small dominance deviation in both groups, which established a similaz parentchild genetic correlation coefficient of 0.44 and 0-46.

5. Under the influence of a new environment, genotypes M M and N N react in different and opposite ways, showing an important fraction (0.246) of the phenotypic variation, which could be attributed to the effects of genes LM and UV.

6. It is concluded that locus MN is a ‘relatively important’ aource of variation of blood pressure in a human population.

Our thanks are due to the Chilean Navy for allowing us to perform our investigation in Easter Island in January-February 1963.

The expedition made use of the technical assistance of Dr C. Guzman, Mr R. Iglesias, Mrs Magdalena Correa, Miss Norma Duriin and Miss Nora Cuevas. We are indebted to Father S. Englert, parson of Hanga Roa, and to Captain J. Martin-Reynolds, governor of Easter Island, for having helped us in field studies; and to Dr E. Covarrubiaa and D. Brncic for in- teresting discussion and advice.

This work was supported by J. Gomez-Millas, Rector of the University of Chile, by the Chilean Navy, and by the Faculty of Medicine of the University of Chile Project 62-12, and the Rockefeller Foundation (Grants 60038 and 63015), in a joint programme.

The Faculty of Medicine gave a further grant ‘Project 63-7.’

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