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Med Clin (Barc). 2009;132(18):709–711
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Nota clı́nica
An update on the molecular analysis of classical galactosaemia patients
diagnosed in Spain and Portugal: 7 new mutations in 17 new families
Laura Gort a,b,1, Ester Quintana a,b,1, Sonia Moliner a,b, Lidia González-Quereda a,
Tania López-Hernández a and Paz Briones a,b,c,
a
Institut de Bioquı́mica Clı́nica, Servei de Bioquı́mica i Genètica Molecular, Hospital Clı́nic, Barcelona, España
CIBER de Enfermedades Raras (CIBERER), Barcelona, España
c
Consejo Superior de Investigaciones Cientı́ficas, Barcelona, España
b
I N F O R M A C I Ó N D E L A R T Í C U L O
A B S T R A C T
Historia del artı́culo:
Recibido el 15 de septiembre de 2008
Aceptado el 5 de noviembre de 2008
Available online 16 de abril de 2009
Background and objectives: Classical galactosaemia is an inherited metabolic disorder due to mutations in
the galactose-1-phosphate uridyltransferase gene (GALT). We previously reported molecular analysis of
83 Spanish and Portuguese unrelated galactosaemic patients. Here we present the results of another
seventeen unreported affected individuals.
Material and methods: DNA from patients was PCR-amplified and sequenced following standard protocols.
Results: Twelve patients diagnosed in Spain were studied. We detected five alleles carrying p.Q188R,
accounting for 21%. Other six alleles (25%) were identified with the mutation p.K285N. We also identified
six novel mutations: p.Q9X, c.328+2T4C, p.I170N, p.C180F, p.V233L and p.P257L. Taking into account all
the Spanish galactosaemic diagnosed patients, mutation p.Q188R is still the most frequent mutation
identified (44.4%). In five new Portuguese patients, five alleles p.Q188R were detected, representing 50%.
One novel mutation (p.F171C) was identified.
Conclusions: Our results confirm our previous observations that p.Q188R is the most frequent mutation in
Iberian Peninsula galactosaemic patients (49%), and that Portuguese and Spanish genotypes differ.
& 2008 Elsevier España, S.L. All rights reserved.
Keywords:
Classical galactosaemia
Mutations
Metabolic disorder
Actualización del análisis molecular de la galactosemia clásica en España
y Portugal: 7 mutaciones nuevas en 17 nuevas familias
R E S U M E N
Palabras clave:
Galactosemia clásica
Mutaciones
Enfermedad metabólica
Fundamento y objetivo: La galactosemia clásica es una enfermedad metabólica hereditaria debida a
mutaciones en el gen de la galactosa-1-fosfato uridiltransferasa (GALT). Nuestro grupo ya publicó los
resultados del análisis molecular de 83 pacientes galactosémicos de España y Portugal. Aquı́ se presentan
los resultados de una nueva serie de pacientes.
Material y método: El ADN de los 17 pacientes estudiados se amplificó por PCR y se secuenció siguiendo
métodos ya descritos.
Resultados: Se analizó a 12 doce pacientes diagnosticados en España y detectamos 5 alelos con la mutación
p.Q188R (21%). Otros 6 alelos (25%) resultaron portadores de la mutación p.K285N. También se
identificaron 6 mutaciones nuevas: p.Q9X, c.328+2T4C, p.I170N, p.C180F, p.V233L y p.P257L. Considerando
a todos los pacientes diagnosticados en España, la mutación p.Q188R sigue siendo la mutación mas
frecuente (44,4%). En 5 pacientes portugueses se detectaron 5 alelos p.Q188R (50%) y se identificó una
nueva mutación (p.F171C).
Conclusiones: Estos resultados confirman las observaciones previas, en las cuales se mostraba que la
mutación más frecuente en la penı́nsula Ibérica es p.Q188R (49%), pero que los genotipos españoles y
portugueses difieren entre sı́.
& 2008 Elsevier España, S.L. Todos los derechos reservados.
Autor para correspondencia.
Correo electrónico: [email protected] (P. Briones).
L. Gort and E. Quintana contributed equally in this work.
1
0025-7753/$ - see front matter & 2008 Elsevier España, S.L. Todos los derechos reservados.
doi:10.1016/j.medcli.2008.11.031
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L. Gort et al. / Med Clin (Barc). 2009;132(18):709–711
Introduction
Classical galactosaemia (MIM 230400) is an inherited autosomal recessive metabolic disorder caused by deficiency of
the enzyme galactose-1-phosphate uridyltransferase (GALT; EC
2.7.7.12). The disease causes neonatal jaundice, cataracts, hepatomegaly, failure to thrive and sepsis. If undiagnosed, it may lead to
a fatal outcome, but the symptoms are reversed with the
introduction of galactose-free diet. However, in spite of good
dietary compliance, long-term outcome can be associated with
growth and mental retardation, verbal dyspraxia, neurological
defects and primary ovarian failure1.
Over 230 different base changes and disease-causing mutations
have been reported in the GALT gene (ARUP galactosaemia database
http://arup.utah.edu/database/galactosemia/GALT_welcome.php)2.
The two most frequent mutations in European populations
are p.Q188R and p.K285N, with frequencies depending on the
population studied. They both have been associated with a severe
form of the disease1,3.
Our previous report on the mutational spectrum of classical
galactosaemia in Spain and Portugal4 analysed 51 Spanish and
32 Portuguese families. Our results indicated a high incidence
of mutation p.Q188R (50% and 57.8%, respectively), and different
frequencies for p.K285N (9.8% in Spanish alleles and absence
in Portugal). In addition, we found a rather high frequency for
p.L195P in Spanish patients (15.7%). Here we present the
molecular results of another 17 unreported affected individuals.
Twelve were from Spain and the other five came from Portugal.
Our aim is to update the spectrum of GALT mutations in the
Iberian Peninsula.
Patients and methods
The patients included were unrelated and came from all the
different regions of Spain and Portugal. In all cases the diagnosis
of galactosaemia was confirmed by reduced or absent GALT
activity in erythrocytes.
Genomic DNA was extracted from patient leukocytes using
standard protocols. Each exon and intron boundaries of the GALT
gene were PCR-amplified. SSCP and RFLP analyses were performed
as described4.
Gene nucleotide numbering was made according to RefSeq
NM_000155.2, with +1 as A of the ATG start codon. The ATG codon
represents +1 for the amino acid numbering according to GALT
protein sequence NP_000146.2.
Results and discussion
Molecular results are shown in Table 1. Twelve Spanish
patients were analysed. We detected five alleles carrying the
frequent mutation p.Q188R, accounting for 21%. Other six alleles
(25%) were identified with the described second most frequent
mutation p.K285N. Remarkably, two patients who were homozygous for this change were of North African origin. We also
identified six novel mutations: p.Q9X (c.25C4T), c.328+2T4C,
p.I170N (c.509T4A), p.C180F (c.539G4T), p.V233L (c.697G4C),
p.P257L (c.770C4T). The four missense mutations change conserved amino acids and they are predicted to affect the protein
function after different prediction programs. The change
c.328+2T4C causes the complete abolition of the donor splice
site in intron 3 after splice site prediction programs and splice site
score calculators; studies on cDNA are under way to elucidate the
effect on the protein.
Table 1
Molecular analyses of the seventeen unreported galactosaemic patients of Spain
and Portugal
Patient
Origin
Allele 1
Allele 2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Portugal
Portugal
Portugal
Portugal
Portugal
p.Q188R
p.Q188R
p.Q188R
p.R148Q
p.R148Q
c.253-2A4G
c.253-2A4G
p.Q9X
p.L195P
p.K285N
p.K285N
c.328+2T4C, p.N314D, p.L218L
p.Q188R
p.Q188R
p.Q188R
p.Q188R
p.S135L
p.Q188R
p.Q188R
p.K285N
p.R148Q
p.K285N
p.V233L
p.S135L
p.I170N
p.P257L
p.K285N
p.K285N
p.C180F
p.Q188R
c.328+33g4a, c.105a4g
p.R333G
p.R204X
p.F171C
In bold, novel changes.
North-African ancestry
Other frequent mutations such as p.L195P and p.R148Q, were
also identified in this patient series. In patient 12, the mutation
c.328+2T4C was present in the Duarte1 variant (p.[N314D; L218L] )
background1,3.
Considering all the 63 Spanish galactosaemic patients diagnosed, p.Q188R is still the most frequent mutation identified
(44.4%). The second most frequent mutation is p.L195P (13.5%)
followed by p.K285N (12.7%). It is worth mentioning that the two
homozygous patients for mutation p.K285N are of Northern Africa
origin. The increase of the immigration experimented in Spain
during the last years is clearly responsible for the change in
mutation frequencies of some inherited diseases such as galactosaemia.
In the five new Portuguese patients, five alleles p.Q188R
were detected, representing 50%. All other changes were only in
one allele each. One novel mutation was identified, p.F171C
(c.512T4G), while mutations p.L195P and p.K285N still remain
undetected in Portuguese patients.
In patient 14 we identified the frequent mutation p.Q188R
in one allele and a combination of two novel changes
([c.328+33g4a; c.*105A4G]) in the other. No other changes were
detected in this patient’s DNA and splicing or transcription sites
prediction programs failed to predict any creation of a new
splicing or transcription factor site for the intronic change. The
variant c.328+33g4a was not identified in 100 control chromosomes, while the change c.*105A4G was detected in one out
of 200 control alleles tested (0.5%). The change c.*105A4G affects
the third 5’ nucleotide of the highly conserved polyadenylation signal AATAAA. Changes in the 3’ regulatory region and,
specifically, in the USS region (upstream region; between the
translational termination codon and the upstream core polyadenylation signal sequence)5, have been described in different genes.
Some of them have been demonstrated to be the cause of a
disease. The effect of these two variants remains unclear. Further
studies must be done to elucidate which of both is the second
disease-causing change in this patient. For the moment, patient
cDNA is not available though its analysis might be very useful.
In the whole group of 37 Portuguese patients analysed to date,
mutation p.Q188R remains the most frequent identified (56.7%).
This is the only frequent mutation as the rest of changes were
found in one or two alleles each.
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L. Gort et al. / Med Clin (Barc). 2009;132(18):709–711
If we take in account all the 100 galactosaemic patients
diagnosed in the Iberian Peninsula, p.Q188R is the most frequent
mutation (49%). Nevertheless, as mentioned before4, the differences observed between the genotypes identified in Portuguese
and Spanish galactosaemic populations continue to be notable.
711
Ministerio de Sanidad y Consumo of Spain and she is a PhD
student of the Department of Biochemistry and Molecular Biology
of the Universitat Autònoma de Barcelona.
References
Acknowledgments
We would like to thank Drs. Castro, Forga, Gacio, Garcı́a,
Garcı́a-Silva, Garcı́a-Valdecasas, Pérez-José, Pujals, Rodrı́gues,
Sousa, Tavares, Ventura, Vilarinho for sending us samples from
their patients.
Financing
The CIBER Enfermedades Raras is an initiative of the ISCIII. Ester
Quintana is a recipient of a fellowship of FIS-ISCIII of the
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