J Egypt Public Health Assoc
Vol. 83 No. 3 & 4, 2008
Detection of Heterozygous Carriers of PKU in
Egypt: Successful Application of a Simple
Biochemical Method
Ebtesam M. Abdalla
Human Genetics Department, Medical Research Institute,
University of Alexandria
ABSTRACT
The absence of a convenient, direct enzymatic assay for detecting
phenylketonuria (PKU) heterozygotes together with the difficulty of
the molecular testing due to the large number of mutations in the
phenylalanine hydroxylase (PAH) gene has resulted in continued
effort to develop an accurate procedure to discriminate the
heterozygous individuals from the homozygous normal population.
Aiming to find out a method that is simple and reliable for PKU
carrier screening, we compared the biochemical data of 20 known
PKU obligate heterozygotes with those of 45 presumed normal
homozygous controls. Fasting blood samples from all subjects were
analyzed for plasma phenylalanine and tyrosine using an amino-acid
analyzer. Micromolar plasma concentrations of phenylalanine and
tyrosine in addition to Phe/Tyr and Phe2/Tyr ratios were determined
and statistical analysis of the difference between the two groups was
done using the student’s t test. Mean values for phenylalanine
concentrations, Phe/Tyr and Phe2/Tyr ratios were significantly higher
in PKU heterozygotes than in control subjects. In addition, ROC
curve analysis was performed for the same four biochemical
variables. The value for the area under the curve (ROCAUC) was
obtained for each parameter with the Phe2/Tyr ratio having an area of
1, which means that it had perfect discrimination. When the ratio
Phe/Tyr was plotted against the Phe2/Tyr, all the studied control
subjects and none of the PKU carriers fell below the values 1.2 and 80,
Corresponding Author:
Dr. Ebtesam Mohamed Abdalla
Human Genetics Department,
Medical Research Institute,
University of Alexandria, Alexandria, Egypt.
Email: [email protected]
J Egypt Public Health Assoc
Vol. 83 No. 3 & 4, 2008
respectively. Finally, by applying the same graphic plot for 20 at-risk
PKU family members asking for premarital carrier testing, nine PKU
heterozygotes were detected. The same results were successfully
reproduced using the values obtained from the ROC curve analysis,
indicating a high degree of accuracy for this screening method. In
conclusion, the used biochemical method is simple and reliable and it
can be useful in the widespread screening for PKU carriers.
Key words: PKU, phenylketonuria, carrier screening, obligate heterozygotes.
INTRODUCTION
Phenylketonuria (PKU) is an autosomal recessive disorder
caused by deficiency of the phenylalanine hydroxylase (PAH)
enzymatic activity as a result of mutations in the PAH gene.(1) It
is one of the most common inborn errors of metabolism, having
an incidence in Caucasian populations of one in 10,000. Based
on this incidence rate, the calculated gene frequency is 0.01 and
approximately two out of every 100 peoples are carriers of PKU.(2)
In general, the PAH mutations that lead to PKU genotypes
result in deficient enzyme activity and reduced stability to
varying extents.(1) Consequently, PAH deficiency presents with a
broad spectrum of clinical and biochemical phenotypes.(3-6)
To
date, more than 500 different disease-causing mutations have
been identified in the PAH gene.(7)
The presence of this large
number of mutations in the PAH gene rendered the molecular
detection of PKU heterozygotes very difficult.
The PAH enzyme, which is the liver enzyme required for
hydroxylation of phenylalanine, exists in a pH-dependent
equilibrium of homotetramers and homodimers.(8) Heterozygote
carriers of PKU do not possess 50% of the normal liver PAH, as
would be expected if the enzyme functioned as a monomer. Thus
the capacity of heterozygotes to metabolize phenylalanine may be
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J Egypt Public Health Assoc
sufficiently
affected
Vol. 83 No. 3 & 4, 2008
to
permit
the
heterozygote
measurement of plasma levels of phenylalanine and
state
by
tyrosine.(2)
The aim of this study was to evaluate the reliability of using
the fasting phenylalanine and tyrosine levels, the Phe/Tyr and
Phe2/Tyr ratios and their graphic plots as a simple method for
detecting PKU heterozygotes that can be subsequently used in
our future carrier screening programs in Egypt.
SUBJECTS AND METHODS
The study was carried out on two separate groups; 20 PKU
obligate heterozygous carriers (9 males and 11 females) and a
control group consisting of 45 presumed homozygous normal
subjects (20 males and 25 females). Obligate heterozygous carriers
were defined as those subjects who had a child with PKU. As for the
presumed homozygous normal controls, they were healthy persons
(children and adults) who were not found to have any family
members with PKU after taking a detailed meticulous family history.
The age of the control subjects was not taken into consideration as it
doesn’t affect the phenylalanine and tyrosine values.(9) None of the
studied females in either group was on oral
birth-control
mediaction or pregnant, since both birth-control mediaction and
pregnancy interfere with heterozygote detection of PKU.(10)
Specimen collection and sample preparation:
Venous blood samples were obtained from all subjects after
an over-night fast.
immediately
after
sulfosalicylic
acid.(11)
Plasma was separated by centrifugation
venipuncture
and
deproteinized
using
The flocculent precipitate was compacted
by centrifugation, and the supernatant fluid withdrawn for
analysis.
Plasma phenylalanine and tyrosine concentrations
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were determined using a Beckman amino acid analyzer.
The
amino acids were quantified as micromoles per litre (µMol/Litre).
Statistical analysis:
•
The biochemical results of the PKU carriers and the control subjects
were compared by means of student’s t test. Four parameters were
determined for each subject; micromolar Phe and Tyr levels,
Phe/Tyr and Phe2/Tyr ratios.
•
The diagnostic performance of these biochemical variables was
evaluated using Receiver Operating Characteristic (ROC) curve
analysis.(12) In a ROC curve the true positive rate (sensitivity) is
plotted in function of the false positive rate (100-specificity) for
different cut-off points of a parameter. The area under the ROC
curve (ROCAUC) which is a measure of how well a parameter can
distinguish between two diagnostic groups was used for
comparison between the four metabolic parameters.
•
Graphic plotting of Phe/Tyr against Phe2/Tyr ratios in PKU
carriers and control subjects were performed as described
previously.(13)
Carrier screening:
In the second stage of the study, we offered the studied
method as a carrier screening technique for the purpose of premarital testing for the members of our recorded PKU families. A
total of 20 at-risk family members (sibs, uncles, aunts and
cousins) asked for testing.
RESULTS
The biochemical data for the 45 presumed homozygous
normal
controls
and
20
PKU
obligate
heterozygotes
are
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Vol. 83 No. 3 & 4, 2008
summarized in Table I.
The mean plasma phenylalanine
concentration, Phe/Tyr and Phe2/Tyr ratios of PKU carriers were
significantly higher than those of the control group. Regarding
the tyrosine levels, no statistically significant difference was
found between the mean values of carriers and controls.
Table (1) : Means, SDs and Ranges of Phenylalanine, Tyrosine, Phe/Tyr
and Phe2/Tyr for Obligate PKU Heterozygotes and Controls.
Biochemical parameter
Control subjects
(n=45)
Obligate heterozygotes
(n=20)
Phenylalanine(Phe)*
µMol/L
61.76 ± 26.82
Range: 12.7-148.5
115.45 ± 39.3
Range: 65.95-214.77
= 0.0001
76.91 ± 38.27
Range: 34.78-193.75
84.0 ± 26.17
Range: 46.4-135.62
= 0.4546
Phe / Tyr*
0.84 ± 0.22
Range: 0.2-1.16
1.4 ± 0.29
Rang: 1.07-1.96
= 0.0001
Phe2 / Tyr*
48.96 ± 20.11
Range: 10.5-77.85
164.95 ± 79.14
Range: 88.89-365.73
= 0.0001
Tyrosine (Tyr)
µMol/L
P. value
* Statistically significant, p<0.05
Despite
these
statistically
different
mean
values
for
phenylalanine between heterozygotes and controls, there was a
substantial overlap between the ranges, rendering phenylalanine
alone useless as a genetic determinant (fig. 1-A). The range of
Phe/Tyr ratio among the controls also overlapped with obligate
heyerozygotes and canceled out this index. (fig. 1-C).
Phe2/Tyr
The
ratio, however, remained free of overlap and seemed to
be an effective discriminant between the control and obligate
heyerozygotes. The highest control Phe2/ Tyr value was 70.05
while the lowest value for PKU carriers was 88.89. (fig. 1-D)
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Vol. 83 No. 3 & 4, 2008
Control
(A)
PKU carriers
Phe level Mol/L
0
50
100
150
200
250
0
50
100
150
200
250
(B)
Tyr level Mol/L
(C)
Phe/Tyr
0
0.5
1
1.5
2
(D)
Phe2/Tyr
0
50 100 150 200 250 300 350 400
Figure (1): The Ranges of: (A) Phenylalanine, (B) Tyrosine, (C) Phe/Tyr
Ratios and (D) Phe2/tyr Ratios of Control Subjects and PKU
Obligate Heterozygotes.
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ROC curve analysis:
After plotting the ROC curves, the value for the ROCAUC was
obtained for each parameter. (fig. 2) It was 0.899 for the
phenylalanine, 0.961 for Phe/Tyr ratio and 1 for the Phe2/Tyr
ratio variable, which means that the last parameter has perfect
discrimination.
The cut-off point 83.37 on the Phe2/Tyr ROC
plot represents 100% sensitivity and 100% specificity.
Phe
Tyr
Phe/Tyr
Phe2/Tyr
Test result variables
Phe
Tyr
Phe/Tyr
2
ROCAUC
0.899
0.633
Standard errora
0.037
0.069
Significanceb
0.000
0.089
0.961
0.021
0.000
0.000
0.000
1.000
Phe /Tyr
a
Under the non-parametric assumption
b
Null hypothesis: true area = 0.5
Figure (2): The ROC Plots and ROCAUC for the Results of the Studied
Biochemical Variables; Phenylalanine and Tyrosine Levels,
and Phe/Tyr and Phe2/tyr Ratios.
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When the micromolar Phe/Tyr ratios was plotted against the
micromolar Phe2/Tyr ratios, all the control subjects had values
within the area bounded by 1.2 and 80 µM respectively. Values
for all PKU carriers were higher for one or both ratios and all of
them fell outside the bounded area (fig. 3). The graphic plot of
Phe/Tyr versus the Phe2/Tyr ratios showed 100% specificity for
the detection of PKU carriers (none of the controls yielded a
false-positive result). Also the sensitivity of the test was 100% as
none of the known PKU heterozygotes showed a false-negative
result.
400
350
2
Phe /Tyr
300
250
200
150
100
Control
Obligate carriers
50
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Phe/Tyr
Figure (3): Graphic Plots of Phe/Tyr Versus Phe2/Tyr Ratios of PKU
Heterozygotes and Controls, According to Hilton et al. (13)
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Screening results:
The plot of Phe/Tyr against Phe2/Tyr, permitted the
detection of 9 heterozygotes among the 20 PKU relatives asking
for premarital carrier testing. Graphic plots of Phe/Tyr versus
Phe2/Tyr for this group are shown in (Fig. 4). The accuracy of
this method was checked by applying the 83.37 cut-off value for
the Phe2/Tyr variable, which is the value obtained from ROC
curve analysis that gives 100% sensitivity and 100% specificity.
All
the
nine
heterozygous
carriers
were
recovered
again
indicating a percentage of correct classification of 100% for the
used method.
400
350
2
Phe /Tyr
300
250
200
150
100
Screening
50
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Phe/Tyr
Figure (4): Graphic Plots of Phe/Tyr Versus Phe2/Tyr Ratios of the
Screened Subjects, According to Hilton et al.(13)
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DISCUSSION
Phenylketonuria (PKU) is a severely handicapping disorder if
not diagnosed and treated early in life.
Its manifestations
include microcephaly, epilepsy, severe mental retardation, and
behavior problems.(14) Screening newborns for PKU is now one of
the most widely used genetic tests in the world, and early
treatment of PKU is the prototype for prevention of a `genetic'
disease.(15) Since the PKU neonatal screening program has not
been implemented in Egypt yet, detection of carriers in PKU
families
followed
by
genetic
counseling
of
heterozygote
individuals may guarantee the prompt detection of PKU in future
children in the family.
However, the PAH, the deficient enzyme in PKU, is almost
exclusively a liver enzyme and any direct assay of its activity
requires a liver biopsy.(2) The mutational analysis of PKU is also
very cumbersome due to the huge number of mutations detected
in the disease gene.(7) For these reasons, indirect methods that
measure plasma concentrations of phenylalanine and tyrosine,
as an expression of PAH metabolizing capacity, have been
developed to discriminate between homozygous normals and
heterozygous controls.(16-18)
In the present study, a biochemical method based on
determination of fasting plasma phenylalanine and tyrosine
concentrations and calculation of Phe/Tyr and Phe2/Tyr ratios,
was used to determine PKU carrier status.
Compared to the
normal homozygous control, the PKU obligate heterozygotes had
significantly higher mean values of phenylalanine, Phe/Tyr and
Phe2/Tyr ratios.
These findings are consistent with those
reported by several studies.(13,19,20)
studies
(13,21),
Also similar to previous
the mean values of tyrosine did not differ between
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carriers and controls in this study.
However, a contradictory
finding was reported by Guldberg et al.
(22)
Despite being significantly higher in PKU carriers compared
to controls, the inter-individual variation of phenylalanine levels
and the Phe/Tyr ratio was extensive in both groups. We were
not able to clearly discriminate carriers who showed values
within the control range.
achieved by the
Phe2/Tyr
However, better discrimination was
ratios for which no overlap was
observed between the obligate heyerozygotes and control ranges.
This finding was reaffirmed by performing ROC curves for
the results of the four studied biochemical parameters. In the
present study, the value of the ROCAUC for the Phe2/Tyr ratio
variable equaled 1, which means that this parameter has perfect
discrimination.
The cut-off point 83.37 on the Phe2/Tyr ROC
plot represents 100% sensitivity and 100% specificity as well.
Lower ROCAUC values were obtained for the other variables. As
ROC curves provide a pure index of accuracy by demonstrating
the limits of a test's ability to discriminate between alternative
states of health over the complete spectrum of operating
conditions, they have been widely accepted as the standard
method for describing and comparing the accuracy of medical
tests.(23)
On the phenylalanine ROC plot, the 65.93 cut-off value gave
100% sensitivity with about 70% specificity, while the 161.95
cut-off value gave 100% specificity (no false-positive results) at
the expense of sensitivity (only 15%). As for the Phe/Tyr ratio
plot the 1.26 cut-off value gave 100% specificity but with 60%
sensitivity. At the 0.99 cut-off value the sensitivity was 100%
with 26.7% false-positive rate. Nevertheless, if a test is used for
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the purpose of screening, then a cut-off value with a higher
sensitivity and minimal false negative rate must be selected.(24)
The proposed carrier screening method used in this study
was the graphic plotting of plasma micromolar Phe/Tyr against
Phe2/Tyr ratios. All the control subjects had values within the
area bounded by 1.2 and 80 µM respectively, while all PKU
heterozygote carriers fell outside the bounded area. In fact, this
method was proposed by Hilton et al.
detecting
PKU
confirmed
by
heterozygotes
many
countries.(18,20,25,26)
other
and
(13)
as a simple method for
its
accuracy
researchers
has
from
been
different
This study reproduces the same results in
the Egyptian population.
The plot of the Phe/Tyr versus Phe2/Tyr ratios also
permitted the detection of 9 heterozygotes among 20 at-risk
family members screened in this study. The accuracy of this
method was checked by applying the results obtained from ROC
curve analysis.
The nine heterozygote subjects were retrieved
again, which adds evidence for the accuracy of the used
screening method.
To our knowledge, despite this multitude of studies done
allover the world to find a simple way for detecting PKU
heterozygotes, no work has been performed on the Egyptian
population.
However,
few
molecular
performed on the PKU patents and
studies
families.(27-29)
have
been
These studies
have confirmed a very high degree of heterogeneity of PAH gene
mutations in Egypt in comparison to other countries.
example, in Denmark
(30)
For
four mutations account for 70% of the
total, while among Egyptian PKU patients, the six most common
Mediterranean mutations represent less than 30%.(29) For that
reason, and also because of its enormous cost, the molecular
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Vol. 83 No. 3 & 4, 2008
screening for PAH carriers in Egypt is not expected to increase
the sensitivity of the carrier screening methodology or the costbenefit ratio, and thus cannot be proposed for clinical use in our
country.
In conclusion, the need for a reliable method to identify
persons carrying PAH mutations is evident. The methods used
in this study offer the PKU families and the population as well an
accurate but more simple test for PKU heterozygote carriers
requiring only a single fasting blood sample.
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