PHENYLKETONURIA

(1)

PHENYLKETONURIA

A

Review

of

Some

Defkits

in Our

Information

David S. Kleinman

Bureau of Maternal and Child Health, California State Department of

Public Health, Berkeley, California

(Submitted June 7; accepted for publication August 15, 1963.)

ADDRESS: 2151 Berkeley Way, Berkeley 4, California.

REVIEW

ARTICLE

123

PEDIAmIc5, January 1964

INTRODUCTORY

REMARKS

P

HENYLKETONU results from a rare but

well-described inborn error of

metab-olism associated with mental retardation.

This error is transmitted genetically as a simple Mendelian recessive autosomal fac-tor.1’ The metabolic defect is the absence or inactivity of the liver enzyme,

phenyl-alanine hydroxylase, which catalyzes the hydmoxylation of phenylalanine to

tymo-sine. ‘ As a consequence, the ingestion of

phenylalanine, which is widely present in food proteins, results in an accumulation of phenylalanine in body fluids and tissues that

interferes with brain function. The level of

serum phenylalanine may rise to 20-30 times

the normal concentrations of 1-3 mg per 100

ml serum phenylalanine. Where renal

func-tions are normal, high levels (about 15-20

mg per 100 ml or more) result in the urinary excretion of phenylpyruvic acid. When phenylketonunic patients ingest low phenyl-alanine diets, the phenylalanine blood levels

may be within or close to the normal

manges.” With early initiation (before 2-3

years of age) and maintenance of such a diet,

mental retardation, neunologic

complica-tions, hyperactivity, eczema, and other

ef-fects of the metabolic defect may be

miti-gated. In general, the earlier the diet is

in-troduced, the more beneficial the effect.s

SCREENING TESTS

Until recently, the only tests which were

cheap and simple enough for screening

large populations for phenylketonunia were

the ferric chloride and related tests (such

as Phenistix) for the presence of phenyl-pyruvic acid in the urine. However, in some individuals who have high blood phenyl-alanine levels, the fenmic chloride urine tests have been negative. Robert Guthnie

me-ported the results of screening 3,118

pa-tients at Newark State School by both the ferric chloride method and tile recently

de-veloped Cuthmie blood inhibition assay. The

screenings were conducted independently.

The fermic chloride screening found 17 phenylketonunics. The Guthnie blood inhibi-tion assay detected these 17 plus 4 others who were missed by the ferric chloride urine test. Of those not detected by the fenric chloride urine test, the blood phenylalanine level of one patient was below 20 mg per

100 ml. In two others, the negative result

was ascribed to difficulties in obtaining urine specimens; the fourth was unex-plained. Centenwall notes that 5-10% of the time, the fermic chloride test may give false negative readings in known

phenylke-tonurics; he advises repeating the test three

times in early infancy (in separate visits) before eliminating the possibility of phenyl-ketonuria. Centerwall et al. report that

test-ing mailed samples of urine-impregnated

filter paper with fermic chloride gives

me-suits which may be unreliable, depending

on the interval between impregnating the filter and the testing.

Armstrong and Low1#{176}report that

phenyl-pyruvic acid is not detectable in the urine

of patients whose serum levels fall below

15-20 mg per 100 ml phenylalanine. Homer

(2)

124

iiot appear in the urine of certain infants

teStedi weekly siilce birth until serum

phenylalanine levels reached 55 mg per 100

nil at 7 weeks in one patient and 65 mg per

100 ml at 6 weeks in another. Thus, the

de-pendence of urinary excretion of

phenyl-pyruvic acid on tile build-up of a high

blood level of phenylalanine and on

matuna-tion of infants’ renal functions and other

pilysiological nlechanisms in the

transarnina-tion and excretion of phenylalanine as

pilenylpyruvic acid may result in an

unde-sirable delay in detecting the metabolic

error.

The Guthnie inhibition assay method,23

using blood from a heel puncture, may make

it possible to avoid the lag involved in the

urine test. The blood is cultured with B.

subtilis in media containing a growth

in-hibiton which inhibition is overcome by

phenylalanine. It is claimed tilat this test

provides quantitative estimates of blood

pilenylalanine levels up to 20 mg per 100

ml.

Bixby, Pallatao, and Pryles used the

Guthnie blood assay to screen 3,222

nesi-dents of two institutions for the mentally

retarded. They found in this group 31 phenyiketonunics. They concluded that the

test is sensitive, reliable, and practical for

screening large populations. They did not

concern themselves, in this study, with the

possible limitations of the procedure in

screening newborn infants in hospital

nurseries, as suggested by Guthnie. It may

take several days for blood phenylalanine

to build tip in tile ile\vbOrn phenylketonuric

ill sufficient concentration for detection;

nlany may 1)e discharged from tile hospital

too early. We should also note that in some

instances, because of inadequate intake of

milk, it may take longer for blood

phenyl-alanine levels to build up. This is especially

true when infants are on mother’s milk or

dilute formula which contains less protein than the usual cow’s milk formula.

Guthnie suggested, with regard to the field trials of ilis procedure, that a filter disc,

impregnated with the newborn infant’s

blood shortly before discharge from the

hos-pital, be tested by the Guthrie assay and that a follow-up cileck on tile blood test be

made by reqtiesting the motiler to send in

a filter disc inlpregnatedl \Vitil the infant’s

urine in 2 to 3 weeks.3

Dr. Robert MacCready, Director,

Diag-nostic Laboratories, Massachusetts

Depart-ment of Public Health, and co-ondinator of

the Guthrie test field trials in that state,

noted, in a letter dated November 21, 1962,

to the Journal of Pediatrics, some difficulty

with the urine test follow-up. Later in

per-sonal comrespondence, he indicated that

the follow-up test of urine-impregnated

flu-ten paper, usiIlg Guthnie inhibition assay,

may not be satisfactory. Indeed, in two out

of the five cases of PKU detected by the

Guthnie blood test and confirmed by LaDu

serum test, the urine test follow-up had

given negative results. Accordingly,

Mac-Cready would prefer that the follow-tip

con-sist of a repeat test of a filter disc

impmeg-nated by the lllfallts blood at 3 weeks of

age.

However, tile latter procedure involves

more inconvenience to tile parent than does

obtaining a urine-impregnated disc and the

parent is Ilot 50 likely to respond to the

follow-up procedure which involves a blood

sample. MacCready also noted that theme

was no follow-up information on tile infants

in the trial \VilO were discharged from the

hospital and tested before 3 to 4 days of age.29 No satisfactory estimate is made of the number of phenylketonunics who may

be missed by this screening program of newborn infants.

Guthnie and Whitney0 report that

ap-proximately 85% of the presumed positives

by the Guthrie inhibition assay blood test, and a much greater percentage of the pre-sumed 1)ositives by the Guthrie inhibition

assay urine test turned out to be false

posi-tives. While many of the urine test false

positives were attributed to “generalized

aminoacidurea,” no definitive information is

presented as to whether the blood test false

positives may be due to transient high

phenylalanine levels, to interference in the

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REVIE\V ARTICLE 125

or combinations of factors. Guthrie and

vIacCready feel tilat the false positives are

a small disadvantage conlpared to tile

bene-fits of early detection of phenviketonurics

who may otilerwise he missed.

EFFECTIVENESS OF EARLY

DIETARY

TREATMENT

Knox presents data on the effectiveness

of early administration of low phenvlalanine

diets ill tile mitigation of mental retardation

associated with phenylketonuria. He

corn-pares I.Q.’s attained by patients with early

and late initiation of diet treatment and also

the treated group with an untreated group;

and finds a negative correlation (in the 43

cases reported) of - .67 between age at

which treatment is initiated and I.Q. sub-sequentiy attained. The slope of the

regnes-sion line is - .45 and is interpreted to

repre-sent the estimated rate of loss of I.Q. with

increased age of initiation of tmeatmellt.

On inspecting the scatter diagran of the

distribution presented Iw Knox, there

ap-iears to he mucil too wide a variation from

the regression line for a correlation as strong

as - .67. Also, tilere is not a constant

nela-tionship along the entire continuum of age

of initiation of treatment and I.Q. attained.

For instance, if the correlation is calculated

for those cases (N = 33) treated 19 weeks

and thereafter, r - .39 and the slope of a

linear regression line is - .36; for those cases

(N = 32) starting treatment at 6 months

and thereafter, r = - .31 and the slope of a

linear regression lines is - .19; for those

cases (N = 31) starting treatment at 7

months and thereafter, r - .24 and the

slope of tile regression line equals - .15;

and finally for those cases (N 29) treated

32 weeks and thereafter, r = - .11 and the

slope is - .07.

2 reports a - .08 to - .15

conrela-tion betiveen age at start of tmeatment and I.Q. in his sample of 26-31 cases and

me-gards this as inconsistent with Knox’s

find-ings. However, Coates’s cases, except for

two patients, were 37 weeks or older at the

start of the diet therapy. On the basis, then,

of our ailalyses above, Coates’s findings

would appear consistent with those of Knox. The strong correlation reported by Knox

over tile range of ages (0-3 years) used

ap-pears to be due to invalid assumptions in

his calculations (linearity of regression and

hornoscedasticity), and to the inordinate

effect of the cases treated very early (before 20 weeks). While there is a marked

nela-tionship between diet initiated very eanl’

in infancy and I.Q. attained, the strong

con-relation does not hold oven the full mange of

cases presented. The regression line is not

linear; there is greater variation in I.Q. at-tamed by patients started on treatment late than by those started early, and the con-relation coefficient is an inappropriate

meas-urement to use except, perhaps, for limited

and specified purposes and over narrow ranges of these observations. The validity of

tile correlation coefficient is contingent on

the assumption that the regression irne is linear throughout the distribution and that

homoscedasticity or uniformity of variation

from the regression line prevails. Knox nec-ognized that the regression is probably not linear, that there is insufficient data to

de-temmme a curve, and notes “there are

reasons for believing the curve may be

sigmoid in shape with an initial flat period

representing relatively little damage during

the first few months followed by a

pre-cipitous fall in the later months of the first

year.”

In addition to tile assumptions noted

above, interpretatioll of such statistical

anal-yses should include consideration of tile

limitations of the measures (I.Q. or D.Q.)

used. A test scone is, by itself, an inadequate characterization of the mentality or

poten-tiality of an individual. Developmental tests

of infants and young children emphasize

sensory and motor capacities while

intelli-gence tests in later childhood involve verbal and conceptual skills. Individual scores may be variable or inconstant and inordinately influenced by innate, developmental, cul-tural, physical, educational, or other

char-actenistics and may not adequately reflect

(4)

x

Age treatment

initiated factors limit not only the diagnostic power

but also tile predictive power of develop-ment and intelligence tests.#{176} To interpret I.Q. alld D.Q. scores adequately,

considena-tion needs to be given to the physical, so-cial, psychiatric, genetic, familial, emo-tional, developmental, and other

deter-minants of behavior.

Nonetheless, it appears appropriate, in

the attempt to assess the role of the factors and configurations of factors in a specific behavior area, to relate the determinants

involved to a limited but central amea or sample of behavior (such as I.Q. test

pen-formance) which is more or less readily scaled and about whose use and limitations there is considerable information. This has

significant methodological aspects and the usefulness of the central measure depends on how vell it serves in prediction and the development of information. Such indices and statistical approaches become the more

necessary as we need to develop our

in-0 In a sample studied longitudinally by Honzik,

MacFariane, and Alien, I.Q’s were reported to

have changed 15 or more I.Q. points for 60%, 20

or more I.Q. loints for 33t, and 30 or more I.Q.

points for 9% of the sample between the ages of 6

and 18 years of age; correlations decreased the

longer the interval between tests, especially

be-tween tests administered at 2 to 5 years of age and

those administered later. This sample had higher

I.Q. scores on the average than norms.3 Charles

re-ported on a series of cases of children deemed to

i)e mentally (leficient on the criteria of school

psy-(liOlO1st evaluation and I.Q. scores below 65-70

points. Many of these children (36% of the group

traced) turned out to he entirely self-supporting.

A more optimistic VieW of the adjustment of this

group was in(licated as compared with original

prognostication.3’ Knobloch and Pasamanick

dis-CUS5 the use of the Geseli developmental

examina-tiOITI and its relationship to predicting later

intellec-tual performance. Findings of correlations of .5 to

.75 between examinations in infancy and tests at

3, 5, and 7 years of age are cited. They note that

the more abnormal the infant, the higher the

cor-relation and state that motor behavior is “not a

determinant of intellectual functioning but rather

of neurologic integrity.”34 Even with such

cor-relations as are cited by Knobloch and Pasamanick

and the connections implied in their statement,

there remains much latitude for individual

varia-tion.

formation from larger numbers of cases as

compared with intensive examinations of

individuals.

The I.Q.’s presented I)y Knox would pro-vide greaten confidence were they obtained

at a later age (6-10 years) when they are more predictive of future performance than those obtained at earlier ages. There are insufficient data to seriously postulate the shape of the curve representing the average decline of I.Q. attained with increasing age of initiation of treatment. But from the data presented by Knox and others, it would not

be unreasonable

to speculate that the curve

may appmoximate a modified rectangular

hyperbola, a sigmoid, or other appropriate shape. For instance:

I.Q.(on the

av-erage) attained

after eatment

Y

(a) Tangents to the curve at particular points

would represent the rate of decline of 1.9.

at that point without treatment (on the

aver-age).

(b) X is age at which appreciable deciine starts.

(c) The curve may involve a very short

seg-ment which is either level or shows a

grad-ual decline for a very short time before the

precipitous decline begins.

(d) Y represents average I.Q. for treatment

started at or close to birth. (Fully protected

against high phenylalanine levels.)

This curve is based on the asumption that the precipitous decline in intelligence

may occur earlier than the “first few months”

mentioned by Knox (above). Alternate

p05-sibilities, not necessarily exclusive of the assumption noted, may be postulated. In-dividuals with this genetic defect may scone lower, on the average, than the population

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REVIEW ARTICLE 127

the high blood phenylalanine levels. This would be consistent with a pleitnopic effect. Better data and more rigorous analyses may provide information in this regard.

One of the impressions, which suggests these assumptions, is that no matter how

early (after allowing a period of time for

diagnoses) diet treatment is initiated in a

PKU, there seems to be some damage. Even among children whose treatment is

initi-ated within several weeks of birth (see Centerwali et al.13 and Homer et al.h1), the children seem to range from normal to low normal to borderline, the distribution being

displaced somewhat to the low side. Center-wall et al.13 state: “It must be concluded

that although mental retardation can be prevented by initiation of a low phenylala-nine diet in early infancy, the diet may not

afford complete protection. . . . A longer follow-up of all these children and others reported in the medical literature will be

necessary to evaluate adequately the final

results of dietary treatment.”

Such curves (I.Q. vs. age of initiation of

treatment) and related considerations are

important in the timing and design of

screening programs and diagnostic proce-dunes to effect maximal and accurate

de-tection with minimal risk of damage. They

may also contribute to our knowledge of

the characteristics and mechanics of the pathological processes. Since we may expect that most of the infant and child cases

which come to attention will be placed on

diet treatment, much of our information will be based on observation of these

pa-tients. The determination of the patterns of response to dietary treatment, including the

description of variations from such patterns, becomes more important in the investiga-tion, development, and evaluation of ex-planations of the disorders involved. In-deed, without an accurate description of the normative mesponse, one could not

de-termine the type and degree of variation extant. We will note, for instance, that, even

with a curvilinear regression line applied to

the data of Knox and others, there would appear to be considerable variation in the

I.Q. after treatment, particularly among those children whose treatment was started late. Consideration of the variability and related factors is an essential component of the explanatory process.

VARIABILITY OF EXPRESSION IN

MENTAL RETARDATION

In the last decade, with the mitigation of

the effects of phenylketonunia through diet-any treatment, there have been more active case-finding efforts and an increase in the number of cases reported outside of insti-tutions. The children found were generally

started on diet treatment as soon as possible. Much of the information regarding the

variability in expressivity of phenylketo-nuria, particularly as it is manifested in mental retardation, comes from our expeni-ence with these children rather than from

institutionalized populations.

Centerwall et cite five

phenylketo-nuric children who were started on diet

treatment at 1-3 years of age and attained

I.Q.

scores after treatment of 52, 60, 73, 80, and 88. All except one showed significant improvement with no apparent correlation between age at which diet was started and final I.Q. or improvement. The one who did not show improvement started with an

I.Q. of 80 when he was placed on the diet at 2% years. The IQ of 80 was maintained although the diet was poorly controlled.

Diedrich and Poser14 describe two chil-dren (siblings), one 4 and the other 3 years old at time of referral for speech therapy

to which they did not at first respond.

It

was discovered that the children were

phenylketonunic and they were placed on

diets. Their I.Q.’s and language develop-ment improved from borderline I.Q.’s

(around 80) to approximately normal (100)

after two years of treatment.

We find, in addition to the differential responses to treatment, several cases who were of relatively high I.Q. prior to duet

treatment and cases who ilave attained “high” I.Q. scones (reported in one instance

(6)

I.Q.

0-20

21-40

41-60

61-80 81+

Knox

#{182}tof 466

64.4

23.2

9.7

1.9

.6

VVright (171(1 Tar/au

I.Q. % of 326 cases

1-20 63

21-50 32

51-70 4

70+ 1

tested at 29 years of age after a younger

sibling was diagnosed as being

phenylke-tonuric. His urine was positive for

phenyl-pynuvic acid by the ferric chloride test but

serum phenylalanine level was only

mod-erately high at 13.2 mg per On the

other hand, retarded phenylketonunics who

are detected and started on low

phenyl-alanine diets at an older age, i.e., after 3

to 4 years of age, do not generally respond

intellectually to treatment. Similar

obsenva-tions have been made of many cllildren

started on treatment from 1-3 years of age.

(

Coates1 presents data in this regard.)

If the data in late-initiated treatment in-dicates anything, it is that there is

con-siderable variability among

phenylketo-nunics at least in their response to treatment.

This variability may reflect, in some degree,

differing individual rates of infliction of

permanent damage on mentality by

abnor-mal metabolites.

The degree of variability in the mentality

attained by untreated phenylketonurics is

more difficult to assess with confidence.

Knox5 and Wright and Tarjan1 have

es-timated the distribution of I.Q. scores

among untreated phenylketonunics to be as

follows:

These findings regarding the distribution of

I.Q. scores among phenylketonunics are

con-sistent with 106 cases surveyed by Paine.17

They are based largely on institutionalized

cases.

The I.Q. scones of institutionalized

phenylketonunics tend to fall considerably

below those of other institutionalized

men-tal netardates. Of the approximately 12,000

patients in California state hospitals for the

mentally retarded in 1960, 32% had I.Q.

scones from 0-19, 35% from 20-39; 24 from

40-59; 8% from 60-79; and 1 had I.Q. scores

of 80 or more.

Knox reviewed tile high-grade cases

me-ported in the literature tip to December,

1959, and found their frequency consistent

with institutional data, and concluded that

only 2-23% of untreated phenylketonumics

are high grade, i.e. , ilave I.Q. scores above

60. This estinlate,

if

valid, would iiidicate

that the variability of I.Q.’s in

phenylketo-nunia is iinlited unless treated early. Knox is

aware tilat tilis estimate ma’ be questioned

and states that “It mtist be asstimed that

re-portage of the tinustial high-grade cases ilas

been at least as complete as the reportage

of tile more tistial mentally defective

in-dividuals \Vitll pilenylketontlnia.’

But while the reportage may be complete,

we cannot be assured that the

ascertain-Illent of cases is as complete for tile

“high-grade” phenylketontinics as it is for the

low-grade or significantly defective

phenylke-tonuric. An evaltiation of the case finding

method is pertinent and essential to tile

determination of tile value of “reportage”

for estimating incidence or other

character-istics of tile poptilation being considered. A

set of observations may be easily biased by

tile method of ascertainment or case finding

tlsed in making these observations. Here

tile method of ascertainment was largely

surveying Patients in hospitals for the

men-tally retarded, and reviewing cases reported

by physicians. Relatively few cases were

initially brought tinder observation

primar-ii)! because tile)’ were recognized as

phenyl-ketonurics. \Vrigllt and Tarjan1 note that

the admission diagnosis for the 21

phenyl-ketonuric patients at Pacific State Hospital

in 1956 was “phenylketontinia” in only three

cases. (The diagnosis of phenylketonuria

was in most cases established later.) The

authors comment: “Most of tile latients

came because their ilyperactive behavior,

irritability, episodes of screaming, noisiness,

uilcontrOllable temper, tailtmulTis , general

tintidness placed a heavy physical and

emo-tional btirden upon the parents and siblings.

Only a few were toilet trained at the time

of admission. In several cases, improvement

was noted after admission suggesting that

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REVIEW ARTICLE 129

aggravated the unacceptable behavior.”

It would be safe to assume that

phenyl-ketonumics who came to the attention of

physicians would be more likely the

seri-ously retarded and troublesome type than

the so-called “high-grade” type who may be

little trouble. The nonsevemely retarded

phenylketonuric, who does not have a

de-tected or an affected sibling is not likely to

be reported, especially if he is older and

thus more likely to escape one of the

screen-ing programs. Therefore, while the

report-age may be as complete for the “high-grade”

cases as for the severely retarded, the case

finding or ascertainment probably is not. On the evidence thus far presented, the distribution of I.Q. scores among

phenyl-ketonumics cannot be given confidently. The bias involved would tend to underestimate

the degree of variability which may exist.

RELATIONSHIP

OF AGE, SERUM

PHENYLALANINE LEVELS,

AND

BRAIN

DAMAGE

Theme is some evidence that the decline of I.Q. with high blood phenylalanine levels

occurs largely in the early years of life.

Homer

et

al.’ describe three phenylke-tonuric children who discontinued the low phenylalanine diet (which was started early in infancy) after 4 years of age without ob-semvabie ill effects. Whether this will be further confirmed has yet to be seen. Cen-terwall et al.b9 advise caution in this regard. Bickel and Grutem report on their experi-ence where a patient whose diet was discon-tinued at 4 years suffered a decline. They

cite other cases in which discontinuation of the diet resulted in declines. However, a personal communication from Armstrong to Bickel describes two favorable outcomes of diet discontinuation in childhood. Bickel and Grutem also reviewed those cases in

which several I.Q. scores were obtained

serially at different ages from untreated

phenylketonunics. It appeared that the I.Q.

of untreated pilenylketontlnics continued to decline until 8-14 years of age but that tile

rate of decline was slowed down

consider-ably in late childhood compared to the

de-dine in the earlier years of cilildhood,

with-out regard to the level of I.Q. involved. For

instance, they cite a child with an I.Q. of 91

at 6 years who at 9 years and without treat-ment, scored 76. They conclude that under the age of 8, untreated patients show a de-dine of I.Q. at D.Q.; that marked decreases

are observed only up to tile age of 8 and

that after age 14 no further decline occurs.

The data thus far obtained do not appear

to have been adequately analyzed (perhaps,

because in their present form, they do not

lend themselves to appropriate analyses). However, Sucil data as are available do indicate that the traumatic effects of high

phenylalanine blood levels are much more

severe in infancy and early childhood than in later years.

Two explanations have been presented for the hypothesis that the detrimental

con-sequences of high phenylalanine levels are

effective largely in infancy and early

child-hood. The first is based on the assumption

that there is a critical period in infancy

and early childhood during which the brain,

because of its rapid development, is

vulner-able to damage connected with the high

phenylalanine levels.

The second hypothesis, perhaps most

definitively stated by Partington and

Lewis,2#{176}is that brain damage is associated with high levels of concentration of phenyl-alanine in the body fluids and tissues, and

that damage occurs in infancy during which

period these levels are highest. Partington

feels that this hypothesis may explain the

variability in intelligence of PKUs and also

the reported instances where a PKU mother

with only moderately high levels of

phenyl-alanine gave birth to a normal child despite

presumed passage of the phenylalanine

across the placenta. An interesting aside

from Partington is the comment that

“mother’s milk” may be better for

phenyl-ketonunics because it has less protetin than

cow’s milk formula. If this were true, then,

we might expect the rate of decline to be

somewhat decelerated in those infants who

are breast fed.

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130

“level of phenylalanine concentration” ex-planation are obviously not mutually ex-clusive. However, some determination of the relative importance and role of each of the proposed processes would be significant

to biochemical and physiological

investiga-tions of the pathology and also to the standards of phenylalanine and protein dep-rivation which are to be incorporated in the diet treatment. This latter consideration is very pertinent in view of the problems involved in the provision and maintenance of the psychological acceptability of the diet and an appropriate nutritional balance.3’

Wright and Tanjan’6 cite an impressive

list of references in support of the assertion that no correlation has been found between the degree of mental defect and the con-centration in the tissues or body fluids of the derivatives of phenylalanine and tymo-sine. These observations may need to be considered in the light of two effects, one statistical and the other developmental (i.e.,

relationship between level in infancy and level at age when observations were made).

If there are considerable variations in the results of analyses on tests due to a lack of

comparability of the quantitative findings of different analytical techniques or to man-dom errors of measurement of phenylala-nine levels, then there will be attenuation of

correlations, i.e., any correlation which ex-ists will be weakened and possibly not

ap-parent. The errors in measurement could be due to either the test technique itself pro-viding variable results (even if valid on the

average) or to variability in the individual due to uncontrolled factors, such as taking fasting and nonfasting serum samples

indis-cniminately. Moncnieff’ states that theme is

no clear connection between degree of

men-tal retardation and blood phenylalanine

levels at the time of examination; but notes that, perhaps, insufficient attention has been paid to the time of taking blood in

me-lation to meals and possible variations

throughout the day in the untreated child.

Armstrong and Low,1#{176}using the

Kapeller-Adler method, find that untreated phenylke-tonumic patients younger than three years

show very much higher levels of serum

phenylalanine (tip to 99 mg per 100 ml)

than do older patients. (They note that while the method was unreliable at lower

levels, it was satisfactory for the higher levels found in most untreated PKU pa-tients.) They also report that electroen-cephalogmaphic abnormalities are more se-veme in younger patients and that two of their atypically “high” I.Q. younger PKU patients had lower serum phenylalanine levels than others in their age group. The data also indicate that the variation of serum

phenylalanine levels among infants is much

greater than that among older patients. Among children less than 3 years old, serum phenylalanine levels ranged from

appmox-imately 25 mg pen 100 ml to almost 100

mg per 100 ml; while among the older pa-tients, the levels ranged from 20 mg per 100 ml to 50 mg pen 100 ml.

Partington and Lewis,2#{176}using the

Uden-friend and Cooper method to determine

plasma phenylalanine levels of 72 untreated phenylketontinic patients varying in age from birth to 54 years, noted that the high-est levels occurred in patients between 11

days and 10 months of age. (Estimates of

the dietary intake of their cases indicated a proportionately higher intake of

phenylala-nine in this age group.) Tile results vary somewhat from those of Armstrong and Low described above in that they found the

levels and variability decreased after 2 to 3

years, while in the group studied by

Part-ington and Lewis the variability and levels decreased after 11 months.

It has been indicated that infancy and

early childhood may be the critical period

for brain damage. Unless a connection is determined between phenylalanine levels in a phenylketonunic’s later years and those

of his earlier years, data obtained in later

years would not rule out the possibility of a

relationship between phenylalanine levels and effects on retardation.

(9)

REVIEW ARTICLE 131

another and 4.5+ mg per 100 ml in a third.

It was noted that several of these infants had been subject to “physiological jaundice of the newborn.” \Vhile Hsia et al.,36 in a

study of 100 term infants, 0-7 days old, did

not find such elevated levels, they did find,

in a study of 48 premature infants 0-21 days

old, several serum pilenylalanine levels

which were moderately elevated. These in-cluded 9 Premature infants with levels

above 6 mg per 100 ml of which 2 were

between 7-7.9, 1 was between 8-8.9 and 1

was above 9 mg per 100 ml. This would

indicate that for whatever reason normal

serum phenylalanine levels may be

some-what frequent. Definitive information as to

the degree and extent of such elevations

would require studies on a larger sample

over a longer age interval. If a large number of cases with transiently elevated blood

phe-nylalanine levels could be identified, it

vould I)e desirable to study the effects, if

ally, of such transient levels.

VARIABILITY

OF EXPRESSION

IN

PHYSIOLOGY AND PATHOLOGY

The relationship between the absence or

inactivity of tile enzyme phenylalanine

hydroxylase and the pattern of genetic

trans-mission has been well established by Jervis and others as is the association between this metabolic error and mental

metarda-3 but the mechanism whereby the

resultant high serum phenylalanine levels

cause the brain damage or even the nature

of the damage involved is not undemstood.’ Abnormal brain myelinization has been ob-served in some necropsies but it was ab-sent in others.25 Sutton’6 discusses some of

the complexities in determining the

mech-anisms involved in the phenotypic

expres-sion of the metabolic error. He notes that phenylalanine is efficiently me-absorbed by

the renal tubules, this being held mespon-sible for the relatively high phenylalanine

levels found in body fluids and tissues of

individuals who cannot metabolize it.

Sut-ton also holds that the high levels may not

in themselves be directly damaging but may

cause the accumulation of other substances

which are more immediately deleterious to neural tissue. He notes that in vitro studies

indicate that some of the aromatic

com-pounds associated with high phenylalanine levels inhibit amino acid decamboxylase me-actions which play an important role in the metabolism of nervous tissue. These

ob-senvations suggest that the mechanisms

through which retardation is affected

in-volve mediating processes, each of which

may be variably affected in different

in-dividuals and at different stages of develop-ment. This is consistent with a finding of variable expression of the manifestation of

phenylketonunia in netirologic pathology,

pigmentation effects, mental retardation, and other channels of somatic expression. Investigations of both the physiological and the biochemical processes involved and their correlates in the various expressions and manifestations are interrelated and should support one another.

ESTIMATES OF POPULATION FREQUENCY

OF PHENYLKETONURIA

One of the earliest estimates of the

fre-quency of phenylketonunia in the

popula-tion was that made by Jervis in 1939.i Jervis examined for phenylketonuria all the

in-mates of 14 state institutions for the

men-tally retarded. The fernic chloride test was

used. Of the 20,300 patients examined, 161 or 0.793% of the inmate population) were

identified as phenylketonumics. In addition,

families of these patients were examined for affected members and 52 close relatives were identified as being affected. The data

were used to establish the unit recessive autosomal chamactem of the gene involved and, incidentally, to estimate the frequency of the character in the population. In this latter regard, Jervis noted : “The frequency

of phenylpyruvic oligophrenia in the

gen-emal population can be determined only on

the basis of the approximate incidence of mental deficiency in the genemal population.

Estimates for this, according to various

authorities, mange from 3 to 0.5. Assuming a

frequency of 1% of which half are in

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PHENYLKETONURIA

the incidence of pilenylpynuvic obigophrenia among institutionalized mental defectives,

the frequency of this condition in the

gen-cral population is of the order of .004%.” If the assumption was made by Jervis

that the institutional population is mepre-sentative of all mental metamdates, the

pmev-alence of PKU should have been calculated

as .00793

x

.01 (if .01 is assumed to be the prevalence of mental retardation in the

community), with a result of .008% rather

than the .004% calculated by Jervis. This would be 1 in 12,500 rather than 1 in 25,000. Unless Jervis assumed that phenylketonumia was found only in institutionalized mental retardates, it would appear that this oft-quoted estimate incorporates an error. In

any case, it is based on inadequately

founded assumptions regarding the fre-quency and definition of mental retardation in the community, the proportion of

men-tally retarded who are institutionalized and

on a comparison of the frequencies of phenylketonunia among institutionalized

and noninstitutionalized mental retandates without establishing a basis for or providing evidence for the validity of such

compani-Sons. His finding of a frequency of .8% for phenyiketonurics among institutionalized retardates is consistent with others. In

Cal-ifomnia in 1961, theme were 89 phenylke-tonunics among the 11,711 patients in state

hospitals for the mentally retarded.18

Armstrong and Low1#{176}found 11

phenylke-tonurics who were born in Utah over a 10-year period in which 224,576 livebirths were recorded. On the basis that not all the children (born with PKU) were detected,

they concluded that the incidents of

phenylketonunia must be somewhat more

than 1 in 20,000.

The true incidence and prevalence would

appear to be higher in view of the bias in both studies which tended toward

enumer-ating only those cases which were

suffi-ciently retarded and living in a home or

community environment in which they

would be considered retarded and referred

for came.

In a British survey of 19,000 children (be-tween ages of 6 weeks and 3 months),27

using a single trial Phenistix test, 1

phenyl-ketonunic was detected; 3 other infants gave

weak reactions. In 2 of these latter cases, it

‘as

determined that, although otherwise developing normally, they were excreting intermittently small quantities of phenyl-alanine in the urine, in one case until 8

months of age. (Serum phenylalanine was not determined but, in view of previous ref-emences, high transient levels of phenyl-alanine may have been present in the blood, even if not in the urine.) In view of the rarity of this disease, the sample was not large enough nor the method rigorous enough to provide confident estimates.

Guthrie and Whitney3#{176} report that in the Guthnie test screening trials approximately

1 in 10,000 infants tested nationally

(includ-ing approximately 1 in 5,000 to 6,000 in-fants tested in Massachusetts) was found to be phenylketonunic confirmed by LaDu serum determinations. This incidence is much greaten than had previously been estimated. The discmepancy may be even greater because the inadequacy of the follow-up procedure (noted earlier) may permit “false negatives” of neonates, with negative blood levels on discharge from the hospital but whose blood phenylalanine level rises later, to go undetected.

CONCLUSIONS

The dietary regimen is a difficult one; it must be closely controlled by the physician if complications are to be avoided.3’ Al-though effective in limiting pathology, if administered early and maintained,

evi-dence is not yet presented as to whether it is fully protective.

Estimates of the incidence and prevalence of phenylketonumia are not well established. Theme are indications that, although rare, the incidence may be considerably higher than the 1 in 20,000 to 25,000 births

pre-viously estimated. Discrepancies in the esti-mates should be explored for possible

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REVIEW ARTICLE ₁₃₃

population frequencies, definitions of

men-tal retardation, and screening and testing procedures.

The degree of variability in the manifes-tations (or expressivity) of phenylketonunia in mental retardation and other effects has not been adequately examined.

Investiga-tion of the factors relating to this variability

may provide information regarding the

me-chanics of pathology and lead to improved

screening and therapeutic measures.

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1964;33;123

Pediatrics

David S. Kleinman