Merit Research Journal of Medicine and Medical Sciences (ISSN: 2354-323X) Vol. 3(9) pp. 427-431, September, 2015
Available online http://www.meritresearchjournals.org/mms/index.htm
Copyright © 2015 Merit Research Journals
Original Research Article
Annual incidence of phenylketonuria in Sulaimani City
Prof. Dr. Adnan M.H. Hamawandi1*, Assist. Prof. Dr. Jamal Ahmed Rashid2,
Heersh H.M.H. Raof Saeed3 and Omed M. Hawrami4
Abstract
1
Department of Pediatrics, School of
Medicine, University of Sulaimani,
Head of Department, Professor in
Pediatrics, Sulaimaniyah, Iraq.
2
Department of Pediatrics, School of
Medicine, University of Sulaimani,
assistant professor in pediatrics,
Sulaimaniyah, Iraq.
3
Department of Pediatrics, School of
Medicine, University of Sulaimani,
Lecturer, Sulaimaniyah, Iraq.
4
Sulaimani Pediatric Teaching
Hospital, Senior House Officer,
Sulaimaniyah, Iraq.
*Corresponding Author’s Email:
[email protected]
Mobile: (+) 964 770 2591646.
Phenylketonuria (PKU) is a hereditary, autosomal recessive disorder
characterized by severe hyperphenylalaninemia which if untreated, would
invariably result in the development of signs and symptoms of classic PKU
with profound intellectual disability. Newborn screening in Sulaimani city
had started in 2013 for screening of (PKU), congenital hypothyroidism and
G6PD deficiency. The purpose of the present study was to find out the
annual incidence of PKU in sulaimani city. All neonates born in sulaimani
city including all hospital deliveries (both private and public governmental
hospitals) in addition to the home deliveries that were referred to
st
(Registration Bureau of Births and Deaths) during the period January 1 to
st
December 31 2014 were included. A heel prick blood sample was taken
from neonates and sent to Sulaimani Pediatric Teaching Hospital laboratory
where phenylalanine level were estimated by ELISA method on the dried
blood spot. From all the screened neonates (8.255cases) who were born in
Sulaimani city, 11 cases were found to have a high level of serum
phenylalanine from the first blood sample and after subsequent serum level
assessment only one case was proven to have phenylketonuria and referred
to nutritional rehabilitation center for therapy and follow up. The annual
incidence of phenylketonuria in Sulaimani city was found to be 1.2 in 10.000
neonates.
Keywords: Phenylalanine, phenylketonuria, incidence, Sulaiamni
INTRODUCTION
PKU was first described by Asbjørn Følling, one of the
first Norwegian physicians to apply chemical methods to
the Study of medicine in 1934 (Gonzalez et al., 2010),
from which eventually came the rationale for early
diagnosis by newborn screening.
Phenylalanine (Phe) is an essential amino acid and
dietary phenylalanine not utilized for protein synthesis is
normally degraded by way of the tyrosine pathway.
Deficiency of the enzyme phenylalanine hydroxylase
(PAH) or of its cofactor tetrahydrobiopterin (BH4) causes
accumulation of phenylalanine in body fluids and in the
brain (Iraj and Melvin, 2011).
Complete enzyme deficiency, in which serum Phe
concentration exceeds 20 mg/dl will cause classical or
severe
PKU (Guttler,
1980),
while
persistent
hyperphenylalaninemia (HPA) is a milder form of the
disorder in which there is only a partial reduction in the
activity of the PAH enzyme from 2 to 5% of normal.
Residual enzyme activity causes mild PKU (Phe
concentration 10 to 20 mg/dl) and HPA ( Phe
concentration 2.5 to 10 mg/dl) (Guttler, 1980; Ushakova
et al., 1997).
The gene coding for PAH enzyme, an enzyme with a
key role in phenylalanine metabolism pathway, is 90 kbp
long and is located on q22–q24.1 regions of chromosome
12. This gene consists of 13 exons and 12 introns
(Williams et al., 2008). More than 400 mutations,
including deletions, insertions, splicing defects, missense
and nonsense mutations, have been identified (National
Institutes of Health Consensus Development Panel,
428 Merit Res. J. Med. Med. Sci.
2000). No single mutation predominates in white
populations, although certain mutations are more
common in specific ethnic groups (Wappner, 2006).
In untreated patients, the hallmark of the disease is
intellectual disability (ID) (Fölling, 1934). Mental
impairment worsens during myelination in early childhood
with increasing dietary exposure, but stabilizes when
brain maturation is complete (Erlandsen and Stevens,
1999). The mechanism by which the elevated
concentration of Phe causes ID is unknown. Excessive
Phe is thought to interfere with brain growth, myelination,
and neurotransmitter synthesis (Paine, 1957).
The early diagnosis of the disease, at least within the
first days of birth, is a critical measure for prevention. In
this regard, neonatal screening has been widely
recognized as a fundamental public health intervention in
the past fifty years (Guthrie and Susi, 1963).
United States Preventive Services Task Force
(USPSTF) recommended neonatal screening as an “A”
category being able to demonstrate to show a high
certainty of benefits (US Preventive Services Task Force,
2008).
PKU is presumed to be more prevalent among the
neonates of consanguineous marriages. High incidence
of relative marriages is directly associated with a high
prevalence of the disease in the population (Nyhan et al.,
2005; Jain et al., 1993).
Because of the often gradual development of clinical
manifestations, HPA is usually diagnosed through mass
screening of newborn infants. In infants with positive
results from the screen for HPA, diagnosis are further
confirmed by quantitative measurement of plasma
phenylalanine concentration. The bacterial inhibition
assay of Guthrie (Andermann et al., 2008), which was the
first method used for this purpose, has been replaced by
more precise and quantitative methods (fluorometric and
tandem mass spectrometry). The method of choice is
tandem mass spectrometry (MS/MS), which identifies all
forms of HPA with a low false-positive rate, and excellent
accuracy and precision. In addition, tandem mass
spectrometry can identify many other inborn errors of
metabolism in a single sample. The national institute of
health (NIH) Consensus Development Conference on
PKU had recommended mutation analysis and genotype
determination on all affected patients for initial diagnosis,
genetic counseling, follow-up, and long-term prognosis
(National Institutes of Health Consensus Development
Panel, 2001).
To the best of our knowledge, the most efficient
prognosis of the disease is to control phenylalaninemia
which should be done before the end of the first month of
birth (Eshraghi et al., 2010). The mainstay of therapy in
PKU is dietary restriction of phenylalanine. This requires
the use of medical foods including phenylalanine-free
protein substitutes (amino acid mixtures or protein
hydrolysates) that supply approximately 75 percent
of protein requirements (except Phe) (MacDonald, 2000).
Treatment should be initiated as soon as possible,
usually before one week of age and Continuation of
dietary restriction throughout life appears to be necessary
for optimal outcomes (Koch et al., 2002; Bosch et al.,
2007). The goals of lifelong nutrition therapy include
normal physical growth and neurocognitive development,
maintenance of adult health, and normal gestational
outcomes in pregnant women with PAH deficiency
(Abadie et al., 2005).
Patient and methods
This retrospective study was done in collaboration with
neonatal screening department in Sulaimani Pediatric
Teaching Hospital that adopted neonatal screening
program for all births inside the city in June, 2013 and as
the first stage for only births inside Sulaimani city and
hoping to expand the program to districts within
Sulaimani governorate in the near future.
All newborns from the city visiting (Registration
Bureau of Births and Deaths), for the purpose of issuing
birth certificate and receiving BCG vaccination were
included. A blood sample was taken through heel prick
and all samples were transferred to Sulaimani Pediatric
Teaching Hospital for neonatal screening department and
screened for phenylketonuria, G6PD deficiency and
congenital hypothyroidism.
In this study the newborns that were referred to
st
(Registration Bureau of Births and Deaths) from (Jan.1 .
to Dec.31st 2014) were included. There were 8.255
newborns. Neonates underwent screening study for HPA
using (quantitative enzymatic assay of Phe by ELISA
method on dried blood spot) within 3 to 10 days after birth
by collecting blood sample from the corner of infant's heel
by experienced technician.
Babies that were excluded in the screening program
included:
1. Sick babies who were asked to postpone their visit to
Registration Bureau.
2. Babies of the refugee families.
3. Babies outside Sulaimani city (i.e districts and towns
outside of Sulaimani governorate) not included.
Blood samples were collected at registration bureau
center on a collection card (Whatman903, where in Iran,
proper citation of manufacture and location) according to
the instruction of manufacturing company, later samples
were transferred to Sulaimani Pediatric Teaching Hospital
/newborn screening department in a special container.
Quantitative enzymatic assay of Phe in dried blood spots
were performed using Neo-PKU Kit, Iran made by
PISHTAZ TEB Company and ELISA plate shaker.
Determination of the Phenylalanine concentration was
done for each sample on standard curve. Results of a
typical standard run of a neonatal Phenylalanine are
shown in table 1 and figure 1.
The cut off value in neonates was 4 mg/dl. For those
Hamawandi et al. 429
Table 1. Concentrations of phenylalanine by optical density.
Figure 1. Calibration curve of phenylalanine
concentration.
samples in which the Phe value was more than 4 mg/dl
concentration the test was repeated in duplicate sample.
If still the readings were still more than 4mg/dl the family
was recalled to Sulaimani Pediatric Teaching Hospital /
newborn screening department and another heel blood
sample collected for reassessment. If the Phe level was
still more than 4mg/dl, then baby/patients was regarded
as having HPA. These cases were referred to the
pediatric dietitian and general pediatric specialist for
further assessment and management as soon as
possible. Because of unavailability of other confirmatory
investigations, 3ml of venous blood sample was taken
and sent to (NeoLab, Athens, Greece), where serum Phe
and Phe/Tyrosine ratio were measured using high
performance liquid chromatography (HPLC) method to
see whether still the levels were above normal to regard
the patient as confirmed PKU. However, patients that
were called back for subsequent level assessment in
hospital and their serum phe level returned to normal
were considered as transient hyperphenylalaninemia.
RESULTS
Out of 8.255 included in this study, the first prick blood
sample analysis showed that 11(%0.133%) of those
neonates had a significantly high level of phenylalanine (
more than 4mg/dl) which required further investigations in
the hospital, 6 of them were females and 5 were males.
Serum phenylalanines of those 11 cases were as follow:
6 cases were between 4.0 and 5.0 mg/dL, 2 cases
between 5.1 and 6.0 mg/dL, 1 case was 7mg/dL and 2
cases were more than 7.1mg/dL.
Parents of all 11 cases were called to come back to
the hospital for further evaluation. On the second
estimation of serum phenylalanine 9 cases were proved
to have normal serum phenylalanine.
Serum phenylalanine of two cases were still high on
the second assessment, one of them was a result of
consanguineous parent while other one have non
consanguineous parent. The one from consanguineous
parent was female with first serum phenylalanine level of
19.1mg/dL and subsequent serum assessment was also
high and confirmed to have phenylketonuria and referred
to nutritional rehabilitation center to start dietary
treatment (Phe free formula which is available in hospital
(PKU1 mix formula, milupa company, turkey made).
While the other one was male from nonconsanguineous parent with a serum phenylalanine level
of 9 gm/dl which had returned to normal before starting
430 Merit Res. J. Med. Med. Sci.
dietary treatment that is why diagnosed as transient
hyperphenylalaninemia.
The annual incidence of PKU in Sulaimani city was
considered to be 1.2 in 10.000 neonates.
DISCUSSION
Screening for congenital metabolic diseases is an
important form of prevention in Pediatrics. This activity is
very useful in the detection of many inborn diseases. It
should be noted that many kinds of congenital defects
can be successfully treated if early detection is made.
Neonatal screening program was implemented in
Sulaimani starting in June 2013. If we compare our
country with many countries at the regional level, we are
behind this practice. Neonatal screening program was
started in California in 1966, the first year for compulsory
newborn screening in California (Peterson et al., 1968),
and in United Kingdom in 1964. To our knowledge,
Sulaimani Pediatric Teaching Hospital was the first in Iraq
to launch this program.
Nutritional rehabilitation center is the only center is
sulaimani city that receives cases diagnosed as
phenylketonuria. During the period of our study
throughout 2014 only one case was registered. The case
was diagnosed by neonatal screening program in
Sulaimani city, this supports that no cases were missed
by our screening program.
Many studies have been done throughout the world to
measure incidence of phenylketonuria in ethnically and
genetically very heterogeneous populations, reflected the
variations of reported PKU incidences (Zschocke et al.,
2003; Jeran et al., 2007; Kremensky et al., 2000).
Unfortunately inside Iraq until 2013 no neonatal
screening program had been launched, in other
governorates in Iraq other than Sulaimani city,
phenylalanine level assessment is done only for diseased
children with suspicion of PKU (Rabab, 2013).
In the regional countries like in Iran, there have been
many studies on the incidence of phenylketonuria; a
study performed by Kabiri and Farhud on 8633 newborns
born in different hospitals in Tehran, the Incidence rate
was calculated as 1.1 in 10.000 (Farhud and Kabiri,
1982), in another study also in Iran the incidence of
(29)
phenylketonuria in Fars province was 1.6 in 10.000,
which is also similar to Farhud study. If we compare both
studies with our results, we see notable similarities, i.e.
an incidence rate of 1.2 in 10.000. Possible explanation
of this similarity is probably because of two reasons, first
we have used machine and kits materials of Iranian origin
and the second reason is probably because of similar
ethnicity of both populations as Fars and Kurdish are
from Iranian descent (Indo-European).if we look at the
data of Asadollah study (Asadollah et al., 2010), we will
see
that
0.04%
of
cases
have
transient
hyperphenylalaninemia which is nearly similar to the
result of this study 0.013% and this support above
reasons. Another reason is that the above mentioned
studies, like this study, considered serum Phe level of
4mg/dl as the cutoff point for referral and further
investigation.
Another important factor that affects incidence rates of
phenylketonuria in a population is the rate of
consanguineous marriage. For example, in the Iran
province of Yazd which is known to have a high rate of
consanguineous marriage, one study shows higher
incidence of phenylketonuria which was estimated to be
1: 5532 (Ordooei et al., 2015). Another example of a
community with a high rate of consanguineous marriage
and high incidence of phenylketonuria is Turkey with
incidence of 1: 2600 (Scriver and Kaufman, 2001). The
incidence of PKU in Europe in 2004, was variable
between countries and the range was between 1:3000
and 1:30000 (Loeber, 2007).
Examples of incidence in few European countries are
like follows: France 1: 13500, United Kingdom 1: 14300,
(32)
Italy 1: 17000 , low incidence of phenylketonuria is
probably because of low consanguineous marriage rate.
ACKNOWLEDGMENT
We like to appreciate the efforts of all staff members of
Registration Bureau of Births and Deaths and all
members of neonatal Screening and nutritional
rehabilitation departments for their help and collaboration.
CONCLUSION
In conclusion the incidence of PKU in Sulaimani city is
relatively high, therefore neonatal screening should be
maintained and extended throughout the region.
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