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Molecular Genetics and Metabolism 77 (2002) 260–266
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Brief Communication
Two novel genetic lesions and a common BH4-responsive
mutation of the PAH gene in Italian patients with
hyperphenylalaninemia
T. Bardelli,a M.A. Donati,a S. Gasperini,a F. Ciani,a F. Belli,a N. Blau,b A. Morrone,a,*
and E. Zammarchia
b
a
Metabolic and Neuromuscular Unit, Department of Pediatrics, University of Florence, Florence, Italy
Division of Clinical Chemistry and Biochemistry, Department of Paediatrics, University ChildrenÕs Hospital, Zurich, Switzerland
Received 30 May 2002; received in revised form 30 July 2002; accepted 31 July 2002
Abstract
Hyperphenylalaninemia (HPA), due to a deficiency of phenylalanine hydroxylase (PAH) enzyme, is caused by mutations in the
PAH gene. Molecular analysis in 23 Italian patients with PAH deficiency identified two novel (P281R, L287V) and 20 previously
described genetic lesions in the PAH gene. The detection of the A403V amino acid substitution in combination with null mutations
in patients with BH4 -responsive PAH deficiency leads us to correlate it with BH4 responsiveness.
Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction
Phenylalanine hydroxylase (PAH, EC 1.14.16.1) deficiency (MIM#261600) is an autosomal recessive disorder
of amino acid metabolism. The disease, characterized by
hyperphenylalaninemia (HPA), has been classified into
three forms: classic phenylketonuria (PKU), variant
PKU, and mild HPA. The main clinical manifestations of
untreated PKU and variant PKU are related to impaired
brain development and include mental retardation, epilepsy, and behaviour problems. Mild HPA, generally
considered a benign disorder, may also present neurological involvement [1,2]. Dietary treatment can prevent
the clinical manifestations of PAH deficiency [3].
Up to now more than 400 different genetic lesions
have been identified in the PAH gene (http//data.mch.mcgill.ca/pahdb_new/). On the basis of the phenotype of homozygous or ‘‘functionally hemizygous’’
patients the majority of PAH mutations has been correlated to a consistent metabolic phenotype [4,5].
Mutations in the PAH gene have also been reported
in patients showing HPA normalization upon oral
loading with the cofactor of PAH, tetrahydrobiopterin
(BH4 ) [1,2,6–9]. This led to the treatment of BH4 -responsive PAH deficiency with BH4 [1,2,9].
Mutations of the PAH gene are heterogeneous in
Southern European populations with marked differences
between regions. Genetic heterogeneity has also been
described in Italian patients with PAH deficiency from
North and South Italy [10].
Up to now no molecular studies have been performed
in patients from Tuscany, Central Italy. In this study, we
characterized the genotype of 23 patients with PAH
deficiency segregating into 21 unrelated families from
Tuscany.
Genetic analysis of the PAH gene, performed by direct sequencing of the patientsÕ genomic DNA, isolated
from lymphocytes, identified two novel transversions
and a common BH4 -responsive mutation.
2. Materials and methods
*
Corresponding author. Fax: +055/570380.
E-mail addresses: malmetab@unifi.it, [email protected] (A.
Morrone).
We studied 23 Italian patients affected by PAH deficiency.
1096-7192/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 1 0 9 6 - 7 1 9 2 ( 0 2 ) 0 0 1 6 6 - X
T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266
Sixteen patients were diagnosed by newborn screening. The other seven patients were born before newborn
screening was introduced and were identified because
of severe mental retardation or delayed psychomotor
development.
Patients were assigned to one of three categories according to pre-treatment blood Phe levels: 13 patients
presented classical PKU with plasma phenylalanine
(Phe) > 1200 lM, four patients showed variant PKU
(plasma Phe 1200–600 lM), and six mild HPA (plasma
Phe < 600 lMÞ (Tables 1 and 2).
In addition, the patients have been grouped into four
subsets according to their metabolic phenotype which is
related to dietary Phe tolerance along the lines of
Guldberg [5]: patients with classic PKU tolerate
<20 mg Phe/kg body weight/day (wt/d) to keep plasma
Phe value < 300 lM; patients with moderate PKU tolerate 20–25 mg Phe/kg body wt/d; patients with mild
PKU tolerate 25–50 mg Phe/kg body wt/d and patients
with mild HPA (MHP) tolerate >50 mg Phe/kg body wt/
d. The patientsÕ metabolic phenotypes are reported in
Tables 1 and 2.
In all patients a BH4 or a combined Phe/BH4 loading
test was performed.
The BH4 loading test was performed with oral administration of 20 mg BH4 /kg body weight and plasma
phenylalanine and tyrosine were monitored at 0 h (before BH4 administration), and at 4, 8 h post BH4 administration.
The combined Phe/BH4 loading test was performed
with oral administration of 100 mg/kg of Phe and, after
3 h, of 20 mg BH4 /kg body weight. In the combined Phe/
BH4 loading test plasma phenylalanine and tyrosine
were monitored at 0 h (before Phe administration) and
at 3, 7, 11, 27 h post Phe administration.
Seven patients (15.1, 16.1, 17.1, 18.1, 19.1, 20.1, and
21.1), diagnosed by newborn screening, with plasma Phe
levels of 1150, 563, 240, 227, 269, 282, and 222 lM, respectively (reference range 40–130 lM) presented a decrease of plasma Phe levels in response to BH4
administration (Table 2).
Patients 15.1, 16.1 (9 and 8 months old, respectively)
had remained on a Phe-restricted diet since the neonatal
period. Phenylalanine tolerance was 36 mg Phe/kg body
wt/d in patient 15.1 and 33 mg Phe/kg body wt/d in patient 16.1.
Patients 17.1, 21.1 (8 and 29 months old, respectively), who were breast fed, were started on a Phe-restricted diet from weaning because their increased
plasma Phe value ð>300 lMÞ. Phenylalanine tolerance
was 33 mg Phe/kg body wt/d in patient 17.1 and
40 mg Phe/kg body wt/d in patient 21.1.
Patients 18.1, 19.1, 20.1 (7, 10, and 18 months old,
respectively) tolerate >50 mg Phe/kg body wt/d to keep
plasma Phe value <300 lM and remains on an unrestricted diet.
261
At present the seven patients with BH4 -responsive
PAH deficiency show a normal psychomotor development and no neurological symptoms.
The patientsÕ genomic DNA was isolated from lymphocytes. PCR amplifications of PAH DNA coding
regions and intron–exon boundaries were carried out.
The direct sequencing of PAH DNA fragments was
performed using Applied Biosystems ABI Prism 310
Genetic Analyser (Foster City, CA, USA).
The patientsÕ genetic lesions and their associated
metabolic phenotype along the lines of Guldberg [5] are
reported in Tables 1 and 2. The designation of the PAH
gene mutations corresponds to that described by Antonarakis [11].
3. Results and discussion
Genetic analysis in 23 Italian patients with PAH deficiency led to the identification of two novel transversions ðc:842C > G; c:859C > GÞ and 20 previously
described mutations in the PAH gene.
The new nucleotide substitution c:842C > G, in exon
7, leads to the novel P281R amino acid change (Fig.
1A). This mutation is located in the CBR2 cofactor
binding region of the catalytic domain and it disrupts
the binding of the PAH enzyme with the BH4 cofactor
[7,12]. Two other amino acid substitutions, P281L and
P281S, were previously reported at the codon P281. The
high frequency of genetic lesions at this codon could be
explained by the presence of a CpG site.
The new transversion c:859C > G, located in exon 8,
leads to the novel L287V mutation (Fig. 1B). The amino
acid Leu287 is located between the residues His285 and
His290, coordinating the PAH enzyme catalytic domain
to a ferric iron, and it is near the amino acid Glu286
which interacts with the BH4 cofactor [7,12]. The L287V
mutation could disrupt the binding of the PAH enzyme
both with the ferric iron and with the BH4 cofactor.
The IVS10-11G > A mutation was detected in eight
patients with classical PKU, combined with one of the
following genetic lesions c.44-45delCT (L15-S16fs),
c.163delT (F55fs), R158Q, R261Q, P281L, P281R, and
IVS11-8G > A (2), respectively (Table 1). The other five
patients with classical PKU were compound heterozygous for: R261X/R261Q (2), S67P/IVS12 + 1G > A,
c.163delT (F55fs)/R158Q, and P281L/L287V (Table 1).
Both the mutations R261X and R261Q, identified in
two brothers, involved the same amino acid located in
the CBR1 cofactor binding region: the R261Q disrupts
the binding of the PAH protein with the BH4 cofactor
and the R261X does not lead to a functional protein
(Fig. 1C). Interestingly, the six patients with classic
PKU carrying mutations located in the cofactor binding
regions of the PAH enzyme (P281L, P281R, and
R261Q) showed no response to BH4 loading.
262
Table 1
Phenotype and genotype in Italian patients with PAH deficiency and without BH4 responsiveness
Patient
Motive of diagnosis
Phe at diagnosis Classification
(n.v. 40–130 lM) at diagnosis
Phe
Metabolic
tolerance
phenotype
(mg Phe/kg
body wt/d)
Genotype
Allele 1
Mental retardation
>1200
Classic PKU
<20
Classic PKU
2.1
>1200
Classic PKU
<20
Classic PKU
>1200
Classic PKU
<20
Classic PKU
4.1
Delayed psychomotor
development
Mental retardation and
autistic behaviour
Mental retardation
>1200
Classic PKU
<20
Classic PKU
5.1
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
6.1
>1200
Classic PKU
<20
Classic PKU
>1200
Classic PKU
<20
Classic PKU
7.2
Mental retardation and
autistic behaviour
Delayed psychomotor
development
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
8.1
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
8.2
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
9.1
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
>1200
Classic PKU
<20
Classic PKU
11.1
Delayed psychomotor
development
Neonatal screening
>1200
Classic PKU
<20
Classic PKU
12.1
Neonatal screening
1080
Variant PKU
13.1
Neonatal screening
780
Variant PKU
14.1
Neonatal screening
950
Variant PKU
28 (at the
age of 20
months)
23 (at the
age of 36
months)
28 (at the
age of 36
months)
3.1
7.1
10.1
Genetic lesion
Associated
metabolic
phenotypes
Genetic lesion
Associated
metabolic
phenotypes
c44-45delCT
(L15/S16fs)
c163 delT
(F55fs)
R158Q
ðc473G > AÞ
R261Q
ðc782G > AÞ
P281L ðc842C > TÞ
Classic PKU
(this work)
Classic PKU
IVS 10-11G > A
Splicing defect
IVS10-11G > A
Splicing defect
IVS 10-11G > A
Splicing defect
IVS 10-11G > A
Splicing defect
IVS10-11G > A
Splicing defect
IVS10-11G > A
Splicing defect
IVS10-11G > A
Splicing defect
IVS10-11G > A
Splicing defect
R261Q
ðc782G > AÞ
R261Q
ðc782G > AÞ
IVS12 þ 1G > A
Splicing defect
R158Q
ðc473G > AÞ
L287V c859 C > G
New mutation
R158Q
ðc473G > AÞ
Classic PKU
Classic, Moderate
PKU
Classic, Moderate
PKU
Classic PKU
Classic PKU
(this work)
Classic PKU
(this work)
Classic PKU
(this work)
Classic PKU
Classic PKU
Classic PKU
Classic PKU
Classic PKU
P281R (c842 C>G)
New mutation
IVS11-8G > A
Splicing defect
IVS11-8G > A
Splicing defect
R261X
ðc781C > TÞ
R261X
ðc781C > TÞ
S67P
ðc199T > CÞ
c163 del T
(F55fs)
P281L ðc842C > TÞ
Classic PKU
Mild PKU
I65T
ðc194T > CÞ
Classic, Moderate,
Mild PKU
Moderate PKU
c280–282 delATC
(Del I94)
?
IVS4 þ 5G > T
Splicing defect
?
Mild PKU
D222G
ðc665A > GÞ
Mild PKU
(this work)
IVS10-11G > A
Splicing defect
Classic PKU
Classic PKU
Classic PKU
(this work)
Classic PKU
Classic PKU
Classic PKU
Classic PKU
Classic, Moderate
PKU
Classic, Moderate
PKU
Classic PKU
Classic, Moderate
PKU
Classic PKU
(this work)
Classic, Moderate
PKU
T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266
1.1
Allele2
Table 2
Phenotype, genotype and BH4 loading test in Italian patients with BH4 -responsive PAH deficiency
Phe at
diagnosis
(n.v.40–
130 lM)
Tyr at
diagnosis
(n.v.50–
140 lM)
Classification
at diagnosis
Phe
Tolerance
(mgPhe/Kg
body wt/d)
Metabolic
phenotype
BH4 loading test
Genotype
Phe (n.v.40–130 lM)
Tyr (n.v.50–140 lM)
Allele1
T0
T4
T8
T0
T8
Genetic lesion
Associated
metabolic
phenotypes
Genetic lesion
Associated
metabolic
phenotypes
Classic,
Moderate,
Mild PKU,
MHP
Mild PKU
and MHP
T4
Allele2
Variant PKU 36 (at the age
of 9 months)
Mild PKU
1150
970
740
78
96
120
R243Q
ðc728G > AÞ
Classic PKU
Y414C
ðc1241A > GÞ
106
Mild HPA
33 (at the age
of 8 months)
Mild PKU
563
306
173
100
102
82
R261Q
ðc782G > AÞ
E390G
ðc1079A > GÞ
270
118
Mild HPA
Mild PKU
240
34
31
84
98
65
18.1
280
138
Mild HPA
MHP
227
76
38
107
146
146
19.1
228
156
Mild HPA
33 (at the age
of 7 months)
>50 (at the age
of 10 months)
>50 (at the age
of 18 months)
MHP
269
78
54
216
179
174
A403V
ðc1208C > TÞ
A403V
ðc1208C > TÞ
A403V
ðc1208C > TÞ
Classic,
ModeratePKU
MHP
15.1
1026
90
16.1
508
17.1
MHP
MHP
IVS10-11G
> A (s.d.)
c1036 delG
(G346fs)
A300S
ðc898G > TÞ
Classic PKU
IVS4 þ 5G
> T (s.d.)
IVS10-11G
> A (s.d.)
?
Classic PKU
MHP
Combined Phe/BH4 loading test
Phe lM (n.v.40–130)
T0
20.1
288
72
Mild HPA
21.1
276
78
Mild HPA
>50 (at the age
of 8 months)
40 (at the age
of 29 months)
Tyr lM (n.v.50–140)
T3
T7
T11
T27
T0
T3
T7
T11
T27
MHP
282
864
582
96
258
66
72
114
120
72
Mild PKU
222
828
684
294
126
54
48
68
72
78
A403V
ðc1208C > TÞ
A403V
ðc1208C > TÞ
MHP
MHP
Classic PKU
T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266
Patient
Phe, Phenylalanine; Tyr, Tyrosine; n.v., normal values; s.d., splicing defect. The BH4-responsive mutations have been underlined. T0 ¼ pre-treatment values; T4,T8 ¼ values detected at 4,8 h post BH4 administration; T3, T7, T11, T27 ¼ values detected at 3, 7,
11, 27 h post Phe administration. In the case of patient 15.1 the full effect of the BH4 administration would probably be at 24 h or later.
263
264
T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266
Fig. 1. 1/A Nucleotide direct sequences of PAH DNA showing the wild-type sequence of exon 7/intron7 boundaries in the normal control and the
new transversion c:842C > G (P281R) at heterozygous state. 1/B Nucleotide direct sequences of PAH DNA showing the wild-type sequence of exon
8 in the normal control and the new transversion c:859C > G (L287V) at heterozygous state. 1/C Nucleotide direct sequences of PAH DNA showing
the wild-type sequence of exon 7 in the normal control and the nucleotide changes c:781C > T (R261X) and c:782G > A (R261Q).
One patient with moderate PKU was compound
heterozygous for c.280-282delATC (delI94)/ IVS4þ
5G > T. Two patients with mild PKU metabolic
phenotype were compound heterozygous for I65T/
R158Q and IVS10-11G > A=D222G, respectively
(Table 1).
T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266
The IVS10-11G > A, IVS12 þ 1G > A, and P281L
mutations have been previously correlated to classic
PKU [5]. Combining our data with that reported in the
literature the c44-45delCT, P281R, IVS11-8G > A,
S67P, and L287V genetic lesions can be associated with
classic PKU and the D222G mutation with the mild
PKU metabolic phenotype (Table 1).
We identified the R243Q/Y414C and the R261Q/
E390G genotypes in two patients with the BH4 -responsive mild PKU metabolic phenotype (Table 2). In
these two patients, the BH4 load caused a significant
decrease in plasma Phe levels. The Y414C mutation has
been previously described at a homozygous level in one
patient with BH4 -responsive PAH deficiency [13] and
the E390G, detected with the null IVS10-11G > A mutation in one BH4 -responsive patient, has been previously correlated to BH4 responsiveness [9].
The other five Italian patients with BH4 -responsive
PAH deficiency were compound heterozygous for the
A403V mutation which was combined with IVS
10-11G > A in two patients, IVS4 þ 5G > T, A300S,
and c.1036delG (G346fsdelG) (Table 2). In these patients plasma Phe concentrations were completely normalized after the BH4 or a combined Phe/BH4 loading
test.
The A403V amino acid substitution has been previously correlated with PAH enzyme residual activity
(32%) and has been described at a heterozygous state in
BH4 -responsive patients [7,12] but has not been correlated with BH4 responsiveness [8]. The detection of the
A403V in combination with the severe genetic lesion
IVS10-11G > A, leading to no functional protein [14],
leads us to associate the A403V mutation with BH4 responsiveness. This mutation is located in a region that
interacts with the secondary structural elements involved in cofactor binding and could result in a mutant
enzyme with a lower binding affinity for BH4 .
In this study the splicing defect IVS10-11G > A was
the most common allele (11/46) among the Tuscan patients with PAH deficiency. This mutation has also been
described as the most frequent PAH gene lesion in
Campania, Sicily, Apulia, and Basilicata [8].
The A403V allele showed a higher frequency (5/46)
than in studies involving patients from North and South
Italy [8]. This genetic lesion can be easily screened for
using BbvI enzymatic restriction analysis. Since in this
study the A403V mutation proved to be common in
Italian patients with BH4 -responsive PAH deficiency we
would like to stress the importance of screening for this
genetic lesion in this phenotype.
Interestingly, the two patients with BH4 -responsive
PAH deficiency and genotype A403V/IVS10-11G > A
showed increased plasma Phe values during weaning
and were started on a Phe-restricted diet.
The patients with BH4 -responsive mild PKU b cdcmetabolic phenotype would probably be the best can-
265
didates for BH4 therapy as an alternative to the
restrictive low-phenylalanine diet.
The neurological involvement recently reported in
patients with BH4 -responsive mild PAH deficiency [1,2]
demonstrates the importance of a BH4 loading test and
an accurate follow up even in patients with this metabolic phenotype.
Acknowledgments
This work was partially supported by grants from the
Association AMMEC and the Azienda Ospedaliera
Meyer, Florence, Italy.
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