GENETICS AND METABOLISM

Presented at the Annual Meeting of the American Academy of Pediatrics, October 6, 1959.

\Vork reported in this paper was done with the aid of grants from the National Heart Institute and the Division of General Medical Sciences, National Institutes of Health.

ADDRESS: Baltimore 5, Maryland.

ARTICLES

565

PEDIATRICS, April 1960

GENETICS

AND

METABOLISM

E. Mead

Johnson

Award

Address

Barton Childs, M.D.

Itarriet Lane I-fume, Jo/ins Hopkins Hospital, and the Department of Pediatrics,

1011135 Hopkinc University Medical School

INTRODUCTORY REMARKS

I am profoundly pleased and grateful to be a recipient of an E. Mead Johnson Award. I am

also pleased to have an opportunity to acknowledge publicly my indebtedness to many

col-leagues, teachers and collaborators whose friendly support through the years has made a career

in pediatrics and genetics possible for me. In particular, I acknowledge an unmeasurable debt

to Dr. Francis F. Schwentker, late Professor of Pediatrics at Johns Hopkins Medical School.

Dr. Schwentker gave me my first opportunities in full-time pediatrics, and his vigorous support

and sympathetic understanding enabled me to studs’ genetics as a Commonwealth Fund Fellow

at the Galton Laboratory, University College, London, under the direction of Professor L. S.

Penrose.

T IS the part of wisdom when about to

._L_

give a dissertation which one hopes will

at once instruct and entertain, to provide at

the outset some definition of the subject

matter. Genetics is the study of the

herita-I)le components of variation; the heritable

factors which determine the range or

ex-tent of diversity. So, genetics is concerned

with heritable differences and likenesses

be-tween individuals and between species.

One emphasizes the differences because one

can be certain of genetic determination of

a particular characteristic only when it

exists ill a population in two or more

alter-native forms. It is the variants which catch

the eye and hold the attention of the

in-vestigator, and which by their presence

suggest more than one form of the gene or

genes which determine that particular

characteristic. I would like in what follows

to present some examples of investigations

of some aspects of genetics in human

popu-lations.

ADRENAL HYPERPLASIA

Several years ago Dr. Melvin Grumbach

and I studied the genetics of adrenal

hyper-plasia, using as our material the patients

of Dr. Lawson Wilkins.1 Since the disease

occurs in more than one member of a

sib-ship and since parents are unaffected, we

suspected that it was genetically determined

and that the affected patient possessed a

double dose of a mutant gene; that is, the

characteristic was recessive. This

supposi-tion was borne out by analysis of the

distri-bution of 76 patients in 56 families; when

the data were statistically processed, the

numbers observed agreed very well with

those expected assuming the recessive

hypothesis. In examining these families we

were struck, as others had been, by the

frequency of affected females as opposed

to males; indeed, there were more than

twice as many females as there were males

(Table I). Genes do not work in a vacuum

but must carry out their function as a part

of the complicated metabolic machinery of

the cell, so that the ultimate measureable

effect of a gene is bound to be conditioned

by the environment in which it is called

Sibs

J’irilism Only

0

0 10 10

Totals

38 4

19

61

* The author is indebted to l)r. .JOIiIi F’. Crigler, Children’s Medical Center, BOStOn, for tile use of data from

families belonging to the Endocrine Clinic of the Children’s Medical Center.

‘I’.BL1 I

RATIo OF :IALI.: PATIF:NT. sirii \IIIIIAZING ADRENAL

IIYI’EIIPLASIA To FF:\IkIF: PATIENTS*

Sex Ratio

M(lles Females Totals SI/F

ItI(IeX CIISCS 3(1 44 114 0.36

Affected sil)s 14 114 32 0.78

‘IOtIIIS 44 10.? 146 0.43

* The author is iIRIehte(1 to I)r. John F. Crigler,

Cliii-(lrens \Ie(li(al Center, Boston, for the use of data froni

faniilies l)elonging to the F.IUli)(rille (Jinie of the

(ihui-(Irens ie(1i(a ICenter.

conditions might I)e sex. So we attempted

to determine whether this apparent

sex-limitation was physiologic or whether it

was due to some bias in the ascertainment

of the cases. We thought it might be

possi-ble that females came more frequently to

the attention of the physician because of

the obvious ambiguity of their sex. So, in

each family we looked at the sex of the

patient who first sought medical care. Now

we see there are nearly three times as many

females as males (Table I). Next, if we

eliminate all the index cases and consider

the sex only of affected sibs, the ratio of

males to females approaches equality. This

suggests that the sex difference is at least

largely due to a bias in the way cases are

detected rather than to sex modification of

gene action, though since there is still a

slight PrePollderallce of females we cannot

ignore this possibility. The implication is

that there must be families containing only

affected males which have not entered

the data.

The genetic analysis suggests that

virihiz-ing adrenal hyperplasia is associated with

a double dose of a mutant gene. We may

use the resolving power of the genetic

analytic method to further define the

con-dition. Several clinical variants of this

syn-drome have been described; some patients

show virihism only, some have hypertension

as well, and others show virilism and

exces-sive salt loss but without hypertension.

Ex-amination of the families revealed that

whatever were the manifestations of the

first case in a family, these were invariably

repeated in subsequent affected sibs (Table

II). We might explain this in one of two

ways. There could be several different kinds

of gene, each capable of occupying the

same site in the same chromosome but each

of which induces a slightly different effect;

one for salt loss, one for hypertension, one

for virilism alone and so on. Alternatively,

each variant of the syndrome might be the

result of genes at quite different loci in

different chromosomes, their ultimate

ex-pression being the result not of variations

on a single biochemical theme but of quite

different events. Though the issue remains

clouded, strong evidence favoring the latter

hypothesis has been given by Bongiovanni

and Eberlein2 who have revealed that the

hypertensive patients have a block in

bio-synthesis of adrenal corticosteroids which is

at variance with that found in the other

types of case. Such disparity could hardly

TABLE II

1!ET EII()G EN EITY BET\V EEN, BUT hOMoGENEITY %\ITIII N, FAMILIES CONTAINING Mon E Tii.&xONF: PATI ENT \ITH \IRILIZI NG ADRENAL HYPEupIASIA*

Type of Disease

Salt losers hypertension \irilisln only

lotais

,“iurnber of

--_________

Index (a.ses Salt Losers Jlyperten.non

0

0

ARTICLES

I)e laid to different representatives of the

genetic material occupying the same locus,

since it is axiomatic that a single locus is

concerned with a single chemical event.

The genetic analytical method has here

shown diversity within a group of persons

suffering rather similar manifestations. This

is all genetic analysis can do; it can provide

a kind of resolution similar to that of a

microscope; it can pose the most pressing

questions; hut it cannot elucidate the modes

of gene expression. For this, one must use

chemical methods.

FAMILIAL NONHEMOLYTIC JAUNDICE

A study of familial nonhemolytic jaundice

carried! omit in collaboration with Drs. James

Sidbury and Claude Migeon may serve as

an example of the chemical approach. This

is a rare dhisease in which tile affected person

l)ecomes jaundlicedi SOOll after birth and

re-11111115 50 for tile duration of life, a period

commonly limited to a few weeks by the

onset and progression of severe neurologic

damage.4 Two of the eight patients who

constitute the total store of this disease at

J

ohns Hopkins have survived. Apart from

their saffron skins and hyperhihirubinemia,

SO average are the measurements of the

various parameters of their development

that they might he taken as fulfilling the

diefinition of the “golden mean.” All eight

of the I)atientS came from four sibships, all

of which were related in various degrees

of intimacy, and the distribution in the

sihships suggested a recessive characteristic.

Nearly all the bilirubin in the blood was

“indirect” and so we were attracted by

the thought that tllese persons might lack

the capability to make “direct” bilirubin;

that is, to conjugate bilirubin with

glu-curollic acid. Indeed such a defect has been

demonstrated in similar patients by

others. Lacking tissues witil which to

test tile activity of tile specific enzyme

con-trolling this reaction, we cllose to administer

substances which are normally excreted in

part as glucuronic acidi conjugates,

meas-tiring ill blood or urine tile extent to which

such conjugates might he made. Among

these were: C14_lahelled cortisol,

tetrally(1r0-cortisone and trichloroethanol. The results

of these tests revealed a severe hut not

corn-p!ete impairment in the conjugating

meclla-nism.

Assuming the jaundiced patients to have

a relatively severe defect, we wished also

to be able to detect the milder defect

pre-dicted for parents and some sibs whom

the genetic analysis stamped as

heterozy-gotes. Sodium saiicylate met our

require-ments of a non-toxic substance which was

excrete(1 in part as a glucuronide and with

little day to day variability. Indeed, tile

consistency with which one individual

makes salicylate glucuronide with

differ-ent doses of the drug, suggests tllat tile

metabolism of this substance is as specific

for the individual as is the nose on his face.

In Table III are shown comparative

excre-tions of salicylate glucuronide for patients,

parents, sibs and grandparents, and for

controls; the values of the parents, all of

whom must be heterozygotes, fall midway

at each dose of the drug between those of

the patients and those of the controls. It

must be said that these patients and sibs

showed neither jaundice nor other obvious

disability. So what these data tell us is

that the same gene produces at once a

characteristic (jaundice) which is perfectly

recessive and a second (a relative deficiency

in glucuronic acid conjugation) which is

in-completely dominant. This is a way of

say-ing that all genes have an effect but often

we lack the means to detect them, and

whether or not we do detect them depends

upon which parameters of their function

we choose to measure.

Figure 1 shows some patterns of excretion

of sahicylate glucuronide by two normal

persons, by the two jaundiced patients, and

by two parents. Of these latter, one (M.C.IT.)

has values falling in tile intermediate range,

the other (M.E.T.), surprisingly, in the

nor-mal range at all doses. Our technique has

not been good enough to detect the gene

effect in this heterozygote. Since we have

measured an effect at some distance

TABLE III

ITIIINAIIY EXCRETION OF SALICYLATE GLUCURONIDE BY AFFEcrED PATIENTS, PARENTS, SIBS, OThERS (ADULT SIBs AND GRANDPARENTS) AND CONTROLS

(Doses are 0.5, 1.0, and 2.0 gm sodium saiicylale.)

0.5 gm

No. Giucuronide

1.0 gm

2(4)

3 27

No.

1 9*

15.5

13.0

Glucuronide

0.5 gm

No. (i’lueuronide

1.0 gm

No. Glucuronide No.

2.0 gin

Parents 6 8.5 8 8.2 7 15.1

Others ‘2 11.2 2 2.2 1 11.3

Controls 13 13.5 20 15.2 5 20.6

(;ilwuronids

24

20

16

4

12

0

8

6 4

2

MCH JOH

MEH

‘

‘ -‘ FIG. 2. Showing segregation of two characteristics

0 5 I 0 2 0 associated with the same gene in the same family.

Fic. 1. Excretions of salicylate glucuronide by pa- (Left) Hemolytic anemia and jaundice. (Right)

GSH-tients, parents and controls. Ordinate represents per stability test. Shaded symbols are reactors, partially cent of urinary sahicylatc which appears as glu- shaded are intermediates and those with crosses cnronicle. Abcissa represents three doses of salicylate not examined. (From Bull. New York Aced. Med.,

administered, in grams. 35:77, 1959, by permission)

Affected Sibs

(‘ont rols

(5) 39

7

30 17.’2

* Values are expressed as per cent of urinary salicylate which appears as the giucuronide.

we must expect the intervention of variables

modifying its ultimate effect. Here there

are questions of absorption, of transport, of

competition with other substances for the

conjugating system, of excretion, and

doubtless of other factors as well.

This variability in expression of genes in

single dose bedevils tile geneticist but is a

26

rule of general application. Sometimes the

measurement of a particular parameter is

such as falls into the range of those of either

of the two homozygotes, and in such

in-stances assignment of actual gene dosage

can be made only by genetic, as opposed to

biochemical, analysis.

NAPHTHALENE-INDUCED HEMOLYSIS

Sometimes the method of analysis of the

distribution of a characteristic among

mem-bers of a family is impossible because the

wrong parameter is studied. An example is

S

0

5

ARTICLES

the i)l1ck square in the left ilaild pedigree

ill tIld’ figure suffered a severe llerflolytic el)iSO(lC, a (liSedSe al)l)arently ullique in this family. As it ilaI)I)eIls tile heniolysis

11a1)hthalene-induced, and the child

PS5(’SS(’(t a sl)cci1l sensitivity to llelllOlysis of: his (‘rvtilrocvtes after ingestion of napll-thll(1l(’ as ell as of certain drugs.

This sensitivity ‘as first StII(lied ill detail

b i grout) at the University of Cilicago

Ui1(Iel’ the dilectioll of Dr. A. S. Alvillg.1 They l)(’gl1l b observing tile effects of

aIlti-1llalarill (Imugs ii P0fl the ervtllrocvte, aiid foulld that

tue

ervthrocytes of only certain Per51l5 \V(1e llehllOlVsed

by

)1inlaqu me and

killdred drugs; tilat these 1)er5Il5 were

Ilearlv all Negroes, and that about 1O of a

ralldlonl saIiI1)le of Negroes were

suscepti-ble. Examination of the erthrocytes of

sellsitive 1)erSnS revealed a deficiency of

glucose-6-)ilosphate dehydrogenase, and

also that the concentration of reduced

glutathione (GSH) in such cells was low.

The latter is in fact consequent upon the

former. Limitation of a characteristic to a

segment of a opulation represents a

chal-lenge to tile geneticist, and when tile trait

is further limited to one ethnic group, the

likelihood of genetic origin is strong indeed.

Dr. \Villiam Zinkham and I, together with

our colleagues in tile Department of

Medi-cine, were stimulated to study this problem

from tile genetic viewpoint.10 We used as

a means of detecting sensitive persons or

“reactors” tile glutathione-stability test

desiglled by Beutler.9 In this test,

erythro-cytes are incubated with

acetyl-phenyl-hydrazine and the GSH content is

meas-ured i)efOre and after the incubation. The

susceptible person in Beutler’s series, which

consisted of males only, was one in whom

tile GSH in whole blood decreased after

incubation to 20 mg/100 ml of packed

erytilrocvtes or less. Using this technique we

examined a group of 328 Negroes including

both males and females, and then tested

the families of all persons designated as

reactors according to Beutler’s criterion. The

family data, involving 32 sibships, were

0

0 75 II

Fic. 3. Frequency distril)ution of values from GSII stal)ihty-test, for male (above) and female (below) fllefllberS Of families. Ordinate: numbers of observa-tions. Abscissa: post-incul)ation values of CSH cx-pressed 111 mg/100 Dli of packed erythrocytes.

(From Bull. Johns Hopkins Hosp., 102:21, 1958, Iw

perniission)

most revealing. It soon became apparent

that males and females were quite different

with respect to this test. This difference is

shown in Figure 3 which depicts the values

for GSH after incubation from members

of the families studied. Clearly tilere are no

occupants of the area between 20 and 40

mg/100 ml among the males, though in the

distribution of values from females this

same range is well filled. Such an

observa-tion suggests that with respect to this test

there are two kinds of male: reactors and

normals, and three kinds of female: reactors,

normals and others showing intermediate

values-that is, between 20 and 40 mgI

100 ml. Now these persons showing these

various responses were distributed over

three generations in some families, which

suggests genetic determination of tile

condi-tion. It was also notable that there was no

charac-teristic, a condition wllich favors strongly

tile location of the gene in tile X

chromo-5011W. Tllis inforiiiation makes intelligible

tile tWo kinds of male alld tile tilree kinds

of female. The former are the two

hemizy-gotes, Illtitdllt and IlorDlal; the latter are

ITItltlIlt h()nlozygotes, heterozygotes and ilormal lR)nlozygotes.

There were, however, certaill exceptions.

Certain mothers \VilO ought to show

inter-IT)e(liate values, in fact, qualified as normal,

as (lid 50fl1C daughters. Here again the test

em1)lOyed ascertains oniy a large

propor-tion, 1)tit Ilot all, of the persons wilicil the

genetic Illetho(l marks a heterozygotes. In

fact, i)ecause tile (listriblition of values

from females is continuous we do not know

Witil any recisi11 just where each of the

three classes ends and the next begins. We

have arl)itrarily broken the curve at 20 and

40 mgIlOO ml because of tile bimodality

sllOWIl in the distribution of values from

males. Genetic analysis lends some validity

to tilis arbitrary measure hut the chemical

metho(I fails with distressing frequency to

surmount the obstacle of misclassification.

An alternative method for detection of

carriers of the gene is assay of the enzyme

glucose-6-phosphate deilydrogenase. This

has been ione by several workers’ with

results whicil suggest its superiority to the

GSH-stahility test. This is not surprising

when one recalls that the low content of

GSH ill the susceptible erythrocyte is a

consequence of the enzyme deficiency.

Measurement of the latter is more proximal

to the gene action.

Tills characteristic has now been studied

i) Iflally investigators in relation to many

hemolvtic anemias induced by many

sub-stances: antimalarials,h) naphthalene,

vita-min K,” Furadantin,” sulfonamides,u) the

fava bean ‘ and others. The discovery of

a common genetic basis for all these

ap-parelltly unrelated disabilities again brings

ilomogeneitv to a heterogeneous syndrome.

The next step in tile further resolution of

tile genetics of this trait will be the detec-tion of variants. Susceptibility is quite corn-ITIOI1 ill 5O1T1 P0Pt1hlti0nS and it is

quite

reasonable to suspect a constellation ol

genes similar to those found among

hemo-globins, the transferrins and the

hapto-globins. Indeed, there is some evidence of

this. The susceptibility has been found in

groups other than the American Negro;

1 Greeks,’ Sephardic Jews,1

and other Mediterranean people have been

found to show it, and the genetic

transmis-sion is the same. But Zinkham has pointed

out tilat it is characteristic of the

erythro-cytes of these latter groups to exhibit a lesser

(or even nil) activity of glucose-6-phosphate

dehydrogenase than those of Negroes,

which nearly always possess some residual

activity. This significant difference is almost

certainly genetically determined, but

pre-cisely how remains to be discovered.

One further point is well illustrated.

Re-turning now to Figure 2 (left pedigree) we

see no familial distribution of the

hemo-lytic anemia but when we consider the

per-formance of the family members in the

GSH-stability test (right pedigree) we see

clearly transmission of the trait through

the family and are able even to detect gene

dosage. Thus what one says a gene does,

depends upon what parameter one measures

and the conditions under which tile

meas-urement is made. One wonders how many

other genetic variants lie hidden behind a

fa#{231}ade of characteristics

which

require the

concatenation of genetic propensities and

unique environmental events.

CONCLUDING COMMENT

I hope that this recital of some

adven-tures in genetics will have thrown into

re-lief one point: the complementarity of

genetics and chemistry! Genetic analysis

brings homogeneity and precision to

con-fusion, but cannot tell us what the genes

do. For answers to these questions we must

use chemical and physical techniques. The

chemical methods can also provide tools

with which to do the genetic analysis. I

be-lieve that the study of the causes of human

diversity has barely begun. Genetics and

biochemistry have much to offer in its

REFERENCES

1. Childs, B., Grumbach, M. M., and Van

Wyk,

J. J.

: Viriiizing adrenal

hyper-Plasia; a genetic aild hormonal study.

J.

Gun. Invest., 35:213, 1956.

2. Bongiovanni, A. M., and Eberlein, W. R.:

Clinical and metabolic variations in the

adrenogenital syndrome. PEDIATRICS, 16:

628, 1955.

3. Childs, B., Sidbury,

J.

B., Jr., and Migeon, C.

J.

: Glucuronic acid conjugation by patiellts with familial nonhemoiytic

jaun-dice and their relatives. PEDIATRICS, 23:

903, 1959.

4. Crigler,

J.

F., and Najjar, V. A. : Familial

nonhernolvtic jaundice with kernicterus. PEDIATRICS, 10:169, 1952.

5. Childs, B., and Najjar, V. A.: Familial

non-hemolytic jaundice with kernicterus; a

report of two cases without neurologic

damage. PEDIATRICS, 18:369, 1956.

6. Arias, I. NI., and London, I. NI.: Bilirubin

glucuronide formation in vitro;

demon-stration of a defect in Gilbert’s disease.

Science, 126:563, 1957.

7. Schmid, R.: Jaundice and bilirubin

metab-olism. Arch. Int. Med., 102:669, 1958.

8. Zinkham, W. H., and Childs, B.: A defect

of glutathione metabolism in erythrocytes

from patients with a

naphthalene-in-duced hemolvtic anemia. PEDIATRICS,

22:461, 1958.

9. Beutler, E.: The hemolvtic effect of

pri-maquine and related compounds. Blood,

14:103, 1959.

10. Childs, B., Zillkham, \V. H., Browne,

E. A., Kimbro, E. L., and Torbert,

J.

V.

A genetic study of a defect in

gluta-thione metabolism of the erythrocyte.

Bull. Johns Hopkins Hosp., 102:21,

1958.

11. Carson, P. E., Flanagan, C. L., Ickes, C. E.,

and Alving, A. S.: Enzymatic deficiency

ill primaqurne-sensitive erythrocvtes. Sci-ence, 124: 484, 1956.

12. Zinkham, W. H., Lenhard, R. E., and

Childs, B. : A deficiency of

glucose-6-phosphate dehydrogenase activity in

ery-throcvtes from patients with fayism.

Bull. Johns Hopkins Hosp., 102:169,

1958.

13. Marks, P. A., and Gross, R. T. :

Drug-in-duced hemolytic anemias and congenital

galactosemia. Bull. New York Acad.

Med., 35:433, 1959.

14. Zinkham, W. H. : An in-vitro abnormality

of glutathione metabolism in erythrocytes

from normal newborns: mechanism and

clinical significance. PEDIATRICS, 23:18, 1959.

15. Kimbro, E. L., Sachs, NI. V., and Torbert,

J.

V.: Mechanism of the hemolvtic

anemia induced by nitrofurantoin. Bull.

Johns Hopkins Hosp., 101:245, 1957.

16. Szeinberg, A., and Sileba, C.: Hemolvtic

trait in oriental Jews connected with an

hereditary enzymatic abnormality of

cry-throcytes. Israel M.

J.,

17:158, 1958.

17. Velltura, S., Grignani, F., and Brunetti, P.:

Premesse per una interpretazion

gene-tica del favismo. Rass. med. Sarda, 60:

459, 1958.

18. Childs, B., and Zinkham, W. H.: The

gene-tics of primaquine sensitivity of the

cry-throcyte, in Ciba Symposium on Human

1960;25;565

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