314 POPESCU ET AL.

HUMAN MUTATION 12:314–319 (1998)

RESEARCH ARTICLE

Mutation Spectrum and Phenylalanine Hydroxylase

RFLP/VNTR Background in 44 Romanian
Phenylketonuric Alleles
Teodora Popescu,1* Michaela Blazkova, Libor Kozak,2 Gheorghe Jebeleanu,1 and
Antonia Popescu3
1
Department of Biochemistry, University of Medicine and Pharmacy, Cluj-Napoca, Romania
2
Department of Biochemical and Molecular Genetics, Research Institute of Child Health, Brno, Czech Republic
3
Department of Paediatrics, University of Medicine and Pharmacy, Cluj-Napoca, Romania

Communicated by Randy Eisensmith

The mutation spectrum and polymorphic haplotype background in 22 Romanian families have
been analysed in this study using the restriction digestion of phenylalanine hydroxylase (PAH)
regions specifically amplified or the DGGE/direct sequencing methods. Eleven PAH mutations
specifically associated with six mutant haplotypes were detected. In spite of the relative heteroge-
neity of the molecular defects in the PAH gene, three mutations covered almost 70% of all alleles:
R408W, 47.72%, 21/44; K363fsdelG 13.63%, 6/44; and P225T 6.81%, 3/44. Among these, R408W,
the most frequent mutation in our population, represented 50% of all the phenylketonuric (PKU)
chromosomes. Splice mutation IVS12nt1g®a affected two PAH alleles (4.54%); the remaining
seven mutations were rare, each having an effect on just one chromosome (1/44), resulting in a
relative frequency of 2.27%. A high frequency was observed in our PKU samples for the relatively
uncommon mutations, K363fsdelG and P225T mutation, suggesting a possible founder effect at
origin. Within the investigated panel, these mutations, both very rare among other Caucasians
were exclusively linked to haplotype 5.8 and 1.7, respectively. These results provide a basis for the
development of a routine molecular analysis of Romanian PKU families. Hum Mutat 12:314–
319, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: PAH; PKU; HPA; Romanian families

INTRODUCTION different PAH mutations in a certain population,

Phenylketonuria (PKU, MIM# 261600), the
most common inborn error of amino acid me-
tabolism, is caused by a high variety of mutations
in the gene for phenylalanine hydroxylase (PAH)
enzyme (E.C. 1.14.16.1). The high clinical and
biochemical heterogeneity observed in this
disease is a consequence of more than 380 dif-
ferent mutations (both expressed and unex-
pressed) identified in the PAH gene. The four
known clinical phenotypes—classical, moderate,
mild, and non-PKU hyperphenylalaninemia
(HPA)—are clearly defined, even though there
are also overlapping intermediary forms among
these phenotypes. The mutation spectrum and
PAH polymorphic background of the PKU alle-
les differ in various populations and geographi-
cal regions. In order to define more precisely the
origin, mechanism, and clinical consequences of
it is necessary to characterise as much of this
population as possible.
The purpose of this study was to describe the
disease-causing mutations and the RFLP/VNTR
associations at the PAH locus in a panel of 22
families from the Transylvanian part of Romania
and to compare these results with the results of
other studies. Using this approach, a more effec-
tive screening program, improved diagnosis, and
earlier implementation of dietary therapy might
be introduced.
Received 18 February 1998; accepted 18 June 1998.
*Correspondence to: Teodora Popescu, Department of Bio-
chemistry, University of Medicine and Pharmacy, Cluj-Napoca,
Romania.
Grant sponsor: Grantova agentura Ceske Republiky; Grant
number: GACR 302/93/2535.

© 1998 WILEY-LISS, INC.


PATIENTS
Twenty-six Romanian children with HPA (44
unrelated mutant alleles) and their parents, when
available, were included in the study. None of the
patients belonged to a consanguineous family. The
patients, including two pairs of univitelline twins
and two sibs, were equally distributed in sexes.
They were of various origins: 18 of Romanian an-
cestry, 5 of Hungarian, one of Gypsy, one of mixed
Hungarian-Gypsy, and one of mixed Ukrainian-
Romanian ancestry. All patients were diagnosed
and followed for diagnosis and treatment of PKU
at the Centre of the third Paediatric Clinic, Cluj-
Napoca, using the Guthrie bacterial inhibition as-
say and high-performance liquid chromatography
(HPLC) measurements of the plasma Phe and Tyr.
The clinical phenotype of the patients was classi-
fied according to the criteria described by Guttler
et al. (1993). They were not tested for BH4 defi-
ciency; therefore, a potential defect in biopterin
metabolism was not excluded. Eleven of the chil-
dren were diagnosed at birth; the other 15 chil-
dren, coming from counties in which PKU
newborn screening is not performed, were later
identified at 9 months to 16 years of age.
Phenotype classification of our probands was
performed, correlating the biochemical parameters
(serum Phe pretreatment value, control Phe values
during the dietary treatment, loading test results)
with the clinical outcome and the specific geno-
type. The effect of mutations was determined when
the predicted PAH residual activity was identified.
METHODS
Genomic DNA was isolated from peripheral
blood leukocytes by standard methods (Lahiri and
Numberger, 1990). More common mutations
known to occur more frequently in our population
(Popescu et al., 1994) were screened by means of
PCR and enzyme digestion, using natural or ampli-
fication created restriction sites. The presence of
the following mutations was tested: R408W, R158Q,
R261Q, IVS12nt1g→a, L48S, G272X, R252W,
I65T, and Y414C. The amplification conditions were
as described elsewhere (Kozak et al., 1995). Remain-
ing unidentified PKU alleles underwent denatur-
ing gradient gel electrophoresis (DGGE); samples
showing altered electrophoretic mobility pattern
were sequenced by direct sequencing.
DGGE
The first 12 exons of the PAH gene with their
intronic junctions in were amplified under condi-
tions described previously (Kozak et al., 1997).
ROMANIAN PKU FAMILIES 315
After generation of heteroduplexes, the amplified
samples were loaded onto 6% polyacrylamide/TAE
gel with 0–9.5 M urea gradient. Electrophoresis
ran in a ProteanRII XiCell apparatus (Bio-Rad) at
170 V, for 4.5 h, in TAE buffer (0.04 M Tris-ac-
etate, 1 mM EDTA, pH 8.0) at a constant tem-
perature of 57°C. Samples with an unusually
migrating band pattern identified by DGGE were
analysed by solid-phase sequencing.
Direct Sequencing
The dideoxy chain termination sequencing
method of Sanger et al. (1977) was carried out
using the Sequenase version 2.0 DNA Sequenc-
ing kit (U.S. Biochemicals) as recommended by
the manufacturer. The sequencing analysis was
performed as described by Kozak et al. (1997).
RFLP/VNTR Haplotype Analysis
Five of the seven diallelic polymorphisms
present in the PAH gene were determined by
digestion of polymerase chain reaction (PCR)-am-
plified DNA with specific restriction endonu-
cleases, as previously described: BglII (Dworniczak
et al., 1991a); PvuII(a) (Dworniczak et al.,1991b);
PvuII(b); MspI (Wedemeyer et al., 1991); XmnI
(Goltsov et al.,1992a). The remaining two diallelic
sites, EcoRI and EcoRV, were examined by South-
ern blot, using a 32P-labelled full-length human
PAH cDNA-phPAH247 (Kwok et al., 1985) as a
hybridization probe (Kozak et al., 1993).
The 30-bp multiallelic minisatellite variable
number of tandem repeats (VNTR) at the 3′ end
of the PAH gene was analysed by PCR according
to the method of Goltsov et al. (1992b).
Restriction fragment-length polymorphism
(RFLP)/VNTR haplotypes were numbered accord-
ing to Eisensmith and Woo (1992).
RESULTS AND DISCUSSION
An 18-year HPA screening in Transylvania,
Romania, demonstrated a PKU incidence of
1:8,000 in newborn and 1:110 in retarded children.
The study of 26 Romanian patients with HPA be-
longing to 22 families identified 11 different PKU
mutations. Results are summarized in Table 1. Five
alleles remain unknown, resulting in 88.64% diag-
nosis efficiency.
As has already been observed by Tyfield et al.
(1997) and Guldberg et al. (1996a), three to six
different mutations are generally responsible for
almost 65% of the mutant alleles. Among these,
one to two mutations represent almost 50% of the
total; the other 25–30% are present, in most cases,
316 POPESCU ET AL.

TABLE 1. Genotype, Biochemical, and Clinical Phenotype for 22 Romanian HPA Patients*
Mutation Phe PAH
(systematic Mutations Silent pretreatment residual
name) (trivial name) polymorphisms Haplotype (µm) Phenotype activity
c.1222C→T/ R408W/ /Q232Q, L385L 2.3/3.8 1920 Classical <1%/<1%
c.1315+1g→a IVS12nt1g→a PKU
c.1222C→T/ R408W/R408W 2.3/2.3 2520 Classical <1%/<1%
c.1222C→T PKU
c.1222C→T/ R408W/K363fsdelG 2.3/5.8 2838 Classical <1%/ND
c.1089delG PKU
c.1222C→T/ R408W/L48S /V245V, Q232Q, 2.3/4.3 1793 Classical <1%/ND
c.143T→C IVS3nt-22t→c PKU
c.842C→T/ND P281L/ND 1.8/2.3 1388 Classical <1%/ND
PKU
c.1222C→T/ R408W/R408W 2.3/2.3 1917 Classical <1%/<1%
c.1222C→T PKU
c.1222C→T/ R408W/R413P /V245V, Q232Q 2.3/4.3 2014 Classical <1%/<3%
c.1238G→C PKU1
c.1222C→T/ R408W/K363fsdelG 2.3/5.8 1428 Moderate <1%/ND
c.1089delG PKU
c.1222C→T/ R408W/R408W 2.3/2.3 1672 Classical <1%/<1%
c.1222C→T PKU
c.1315+1g→a/ IVS12nt1g→a/ Q232Q, L385L/ 3.8/5.8 1789 Classical <1%/ND
c.1089delG K363fsdelG PKU
c.1222C→T/ R408W/R261Q 2.3/1.8 1079 Classical <1%/30%
c.782G→A PKU
c.1222C→T/ R408W/R408W 2.3/2.3 ND Classical <1%/<1%
c.1222C→T PKU
c.533A→G/ E178G/R252W /L385L 1.7/69.3 1515 Non-PKU ND/<1%
c.754C→T HPA
c.1222C→T/ R408W/R408W 2.3/2.3 1678 Classical <1%/<1%
c.1222C→T PKU
c.1222C→T/ R408W/P225T 2.3/1.7 1243 Classical <1%/ND
c.673C→A PKU
c.1089delG/ND K363fsdelG/ND 5.8/1.8 1600 Classical ND/ND
PKU
C.1222C→T/ND R408W/ND /V245V, Q232Q, 2.3/4.3 1538 Classical <1%/ND
IVS3nt-22t→c PKU
c.1222C→T/ R408W/P225T 2.3/1.7 ND Classical <1%/ND
c.673C→A PKU
ND/ND ND/ND V245V, Q232Q, 4.3/4.3 656 Classical ND/ND
IVS3nt-22t→c/ PKU
V245V, Q232Q,
IVS3nt-22t→c
c.1222C→T/ R408W/E390G /V245V, Q232Q, 2.3/4.3 984 Non-PKU <1%/ND
c.1169A→G IVS3nt-22t→c HPA
c.1089delG/ K363fsdelG/K363fsdelG 5.8/5.8 3319 Classical ND/ND
c.1089delG PKU
c.1222C→T/ R408W/P225T 2.3/1.7 1515 Classical <1%/ND
c.673C→A PKU
PKU, phenylketonuria.
*Frequencies of single mutations: R408W 47.72%(21/44); K363fsdelG 13.63%(6/44); P225T 6.81%(3/44); IVS12nt1g→a
4.54%(2/44); and L48S, P281L, R413P, R261Q, E178G, R252W, E390G 2.27%(1/44).

in just one or two alleles. This is in accordance
with our findings of mutation frequency (Table 1).
Using the methods described above, both mu-
tant alleles were identified in 18 patients. Among
these, 72.72% (16/22) are compound heterozygotes
for two different mutations, and 27.2% (6/22) are
homozygotes for R408W or K363fsdelG. In three
patients, only one mutant allele was identified; in
one subject, no abnormal electrophoretic pattern
was observed in the first 12 exons of the PAH gene
with their splice junctions. There are two possible
explanations for this. The disease-causing muta-
tion could be either in exon 13 of PAH gene or
deep in the intronic sequence; or, as might be the
case for the patient with no mutation detected, it
could be caused by BH4 HPA deficiency, especially
when the low pretreatment Phe value of 656 µmol/
L, the normal DGGE aspect, and the very severe
clinical phenotype are taken into account.
The mutations detected were predominantly
distributed in exons 6–12 (38/44, 86.36%). The
most frequently affected (50% of disease-causing

mutations) was exon 12 (R408W, R413P). Exon
7, which is known to be the most hypermutable
exon of the PAH gene (Kleiman et al., 1992), had
the most various mutations (R252W, P281L,
R261Q). Five of the 11 mutations (R408W,
R261Q, P281L, R413P, R252W) involved a CpG
hotspot, suggesting recurrent mutation as a mecha-
nism of origin. Another mechanism, explaining the
occurrence of mutations P225T and K363fsdelG,
is the founder effect; these mutations have high
frequency and are linked with specific haplotypes
in studied population.
The most frequent mutation responsible for
PKU in the Romanian population is R408W, ac-
counting for almost 50% of all mutant alleles.
Eleven patients (68.75%) were heterozygous, and
five were homozygous for R408W. The mutation
was always associated with haplotype 2.3 (H2.3)
alleles, according to our results. This finding con-
firms the association of the mutation with H2.3
PKU alleles in Central and Eastern Europe, where
it is considered to be of Balto-Slavic origin (Konecki
et al., 1991; Eisensmith et al., 1995). Even though
the Slavic origins are lacking in the Romanian PKU
population, frequent Slavic migrations probably foot-
printed the genetic apparatus of Romanian ethnical
group (Comsa et al., 1987; Heitel et al., 1994; Horedt
et al., 1982, 1986).
The second most frequent mutation was found
to be frameshift microdeletion K363fsdelG
(Table1). This deletion causes premature termi-
nation of the protein chain eliminating exons 13,
12 and partly 11. These exons encode the cata-
lytic center; thus, inactive enzyme is probably
produced. One patient homozygous for this mu-
tation and four compound heterozygotes, bear-
ing null mutation on the other PKU chromosome,
sustained the severe classical clinical phenotype,
suggesting the severity of this mutation. Further
in vitro expression studies of the mutant PAH
enzyme are necessary to confirm this hypothesis.
The deletion is flanked by short repeated se-
quences (5′AGAAG, 3′AGAAG); thus, it can be
assumed that DNA strands caused slipping of
polymerase during DNA replication, thus en-
abling mutation at this site (Jaruzelska et al.,
1992; Kleiman et al., 1992).
The third most frequent mutation in our study
was P225T (Table 1). This mutation is found only
in the Romanian (H1.7) and Czech (H1.9) PKU
population (Hoang et al., 1986; Kozak et al., 1997).
Association of one mutation with different num-
bers of VNTR is not unusual; it can be explained
by the high mutational rate of such polymorphisms
ROMANIAN PKU FAMILIES 317
(Goltsov et al., 1992a,b). Two mutations specific
for very mild non-PKU HPA (E390G and E178G)
were identified in our studied samples. In both
cases, null mutation (R408W and R252W, respec-
tively), was present on the other PAH allele. These
two non-PKU HPA mutations were also observed
in other very mild HPAs combined with severe
mutations; however, these were different from
those in our patients (IVS12nt1g→a and IVS10nt-
11g→a, R408W, respectively). Our findings con-
firm previous observations (Desviat et al., 1987;
Economou-Petersen et al., 1992; Guldberg et al.,
1994; Hoang et al., 1986) that the combination of
severe and mild PKU mutation results in mild
HPA, rather than classical PKU.
The distribution of PKU mutations in Roma-
nian patients included in this study is very similar
to that observed in other Caucasian populations
(Slavic, Scandinavian, or Mediterranean). How-
ever, mutation of Asian origin (R413P) is present
as well. It has been speculated (Eisensmith et al.,
1995; Konecki et al., 1991) as to the means of
spreading different mutations in distinct ethnic
groups, e.g., direct or intermediate genetic passage
by different population migrations in history. Thus,
Mediterranean mutations in Romanian population
are connected with the Daco-Roman origin of the
population (Comsa et al., 1987; Heitel et al., 1994,
Horedt et al., 1986), while Scandinavian and
Slavic mutations arrived probably by the way of
multiple Germanic and Slavic migrations (Heitel
et al., 1994; Horedt et al., 1986, 1982).
The distribution of mutant haplotypes at the PAH
locus in 44 alleles investigated was as follows: H2.3
(22/44, 50%), H5.8 (6/44, 13.6%), H4.3 (6/44,
13.6%), H1.7 (4/44, 9.09%), H1.8 (3/44, 6.81%),
H3.8 (2/44, 4.54%), and H69.3 (1/44, 2.27%).
H2.3 was predominant in our study, in agree-
ment with other Central and Eastern European
populations. H2.3 was found to be associated with
severe R408W mutation (21/22, 95.45%) and with
unknown mutations (1/22, 4.54%) in patients with
classical PKU phenotype. The next most frequent
was H1.7 or H1.8 (H1, 5/44, 15.90%), on which
four different mutations (P225T, E178G, R261Q,
P281L) and one unknown mutation were found.
H5.8, not frequent in other populations, was in
our study in strict linkage disequilibrium with the
frame shift deletion K363fsdelG. H4, which is to-
gether with H1 relatively common in both normal
and mutant PAH alleles (Konecki et al., 1991) was
associated with various mutations (L48S, R413P,
E390G), as well as with three unknown mutations.
This finding confirms the previously established
318 POPESCU ET AL.
finding that, unlike H2 and H3, H1 and H4 are
generally associated with many different mutations.
H69.3, which is quite unusual, was present in our
study with the R252W Gypsy PAH allele.
Four different polymorphisms (V245V, Q232Q,
L385L, IVS3nt-22-t→c) in coding part of the PAH
gene, specifically associated with mutated H4.3,
H3.8, and H69.3 alleles, have been found in some
of the patients studied. The identification of these
silent mutations on new PAH alleles will predict
with high probability the presence of haplotypes
4.3 or 3.8 and of mutations R413P, L48S,
IVS12nt1g→a, and E390G.
Taking into account the high mutational het-
erogeneity at the PAH locus and the specific mu-
tation/haplotype associations in distinct ethnical
groups, molecular analysis covering most popula-
tions aids in elucidating the origin, spread, and
mechanism of origin of these mutations.
Nineteen PKU patients had the classical phe-
notype; only one was a moderate HPA form, and
two had HPA non-PKU. The severe classical phe-
notype requiring strict dietary regimen was pre-
dominant in our cases (86.36%). The combined
study of mutations (genotype) and clinical and
biochemical phenotype (PAH residual activity,
pretreatment and Phe value during treatment, Phe
loading test when performed) demonstrated gen-
erally a good genotype–phenotype correlation. Two
exceptions to the rule were found: a case of non-
PKU HPA (E178G/R252W) with high pretreat-
ment Phe level (1,515 µmol/L), but with 0.1 g/kg
by weight loading test eliminated within 24 h, mini-
mal neurological changes, and normal IQ, and a
case of moderate HPA diagnosed at 16 years of
age because of the patient’s bad school results. This
patient had a very high pretreatment Phe value
and a genotype (R408W/K363fsdelG) correspond-
ing more to the classical phenotype.
Genetic analysis performed in our study of Ro-
manian PKU patients is important from the point
of view of diagnosis confirmation, for prognosis and
by those correct and earlier optimal dietary therapy
will be implemented. Proper genetic counselling
might then be introduced, with the possibility of
detecting carriers in families at risk.
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