2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.064
isolation and characterization of a novel cHO cell mutant
defective in peroxisome biogenesis factor, PEX , containing
aberrant huge peroxisomes
K. Ghaedi 1,2,3 , Y. Fujiki 4,5 ;
1 Biology Dept., Isfahan, Islamic Republic of Iran, 2 Royan Institute,Isfahan Research
campus, Isfahan, Islamic Republic of Iran, 3 Biology Dept, Faculty of sciences,
Kyushu University, Fukuoka, Japan, 4 Biology Dept., Faculty of Sciences,
Kyushu University, Fukuoka, Japan, 5 JST, SORST, Tokyo, Japan.
Peroxsiomal Biogenesis Disorders (PBDs) are characterized as the
congenital cerebro-hepato-renal disorders which are mostly due to the
defects in the biogenesis of peroxisomes . In order to study molecular
mechanisms of peroxisome biogenesis, CHO-K1 cells have been
widely used by our group as the best model cells . Using CHO-K1 cells
stably transformed dually with PEX2 (to prevent frequent isolation of
pex2 mutant cells) and EGFP fused downstream of N-terminally 40
amino acid residues of Pex3p, termed TkaEG3-40 cells, we have started
a procedure of mutagenizing with MNNG as a chemical mutagenic
component and following culture in the presence of P9OH with subsequently
an exposure to UV . By the above approach, we have isolated
several CHO cell mutant cell lines defective in peroxisome biogenesis
which were resemble to the patients’ firoblasts of the PBDs. One of
CHO cell mutant colonies which, showed to have morphologic aberrant
huge peroxisomes but very few in numbers, termed ZPEG301
cells . ZPEG301 cells belonged to the PEX16 complementation group
as numerous punctuate structures of peroxisomes were restored after
its cDNA transfection in to those cells . Moreover temperature shift assay
to permissive temperature at 30 degrees of centigrade showed a
moderate increase in peroxisomal structures in ZPEG301 cells which
is presumably due to the stabilization of peroxisomal membrane proteins
such as PMP70 . Biochemical pattern of peroxisomal proteins
have documented our hypothesis . Taken together these results demonstrate
that Pex16p is involved in morphological control and division
of peroxisome in mammals besides of its main role in peroxisomal
biogenesis .
P01.065
molecular diagnostics of phenylketonuria
E. Polák1 , A. Ficek1 , L. Kádaši2 ;
1Comenius University, Faculty of Natural Sciences, Department of Molecular
Biology, Bratislava, Slovakia, 2Institute of Molecular Physiology and Genetics,
Slovak Academy of Science, Bratislava, Slovakia.
Phenylketonuria (PKU) is an autosomal recessive inherited disorder
arising from the deficiency of phenylalanine hydroxylase (PAH), which
catalyses the essential convertion of phenylalanine (Phe) to tyrosine
(Tyr) . In the majority of cases, PKU is caused by mutations in the PAH
gene, and it presents with different phenotypes which are classified
according to Phe tolerance . More than 500 mutations have been described
world-wide and the PAH enzyme has been fully characterized .
The incidence of the disease in the Slovak population had been estimated
to be 1:10 000 newborn . Seven causative mutations which have
been so far identified make up 70 % of all PKU alleles. Thus the aim of
our work was to carry out a complete mutation analysis in a sample of
48 unrelated PKU patients with 1 or with no known mutation . The DH-
PLC (Denaturating High Performance Liquid Chromatography) method
was applied for screening all 13 coding exons of the PAH gene . Identified
heterozygous fragments were subsequently sequenced to characterize
the DNA variants . Sequencing revealed 16 mutations (of which
1 is new) not previously found in the Slovak population, and 2 frequent
polymorphisms in the coding region of the PAH gene . Corresponding
frequencies for all mutations were estimated, and the classification according
to phenotypic categories of PKU was identified.
P01.066
Assessment of the severity of the PAH gene mutations detected
in serbian population: genotype-phenotype correlation and
functional studies in vitro
M. Stojiljkovic1 , B. Perez2 , L. R. Desviat2 , C. Aguado2 , M. Ugarte2 , S. Pavlovic1 ;
1Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia,
2Centro de Biología Molecular Severo Ochoa, Madrid, Spain.
Phenylketonuria (PKU) is caused by deficiency of hepatic enzyme,
phenylalanine hydroxylase (PAH) . Although numerous factors contribute
to the PKU phenotype, several studies showed that mutations in
the PAH gene are main determinant of PKU severity . In the study on
34 unrelated patients with phenylketonuria from Serbia, we identified
19 disease-causing mutations: L48S, R408W, P281L, E390G, R261Q,
R158Q, I306V, IVS12+1G>A, Q20X, R111X, V177L, P225T, R261X,
L15/S16fsCTdel, S231F, R252Q, R297H, IVS10-11G>A and R413P .
According to pretreatment serum phenylalanine level, patients were
assigned to classic PKU (65%), mild PKU (35%) and MHP (0%) . The
most frequent mutation in Serbia is L48S . Since genotype-phenotype
correlation inconsistency for L48S mutation has been previously reported,
we investigated genotypes involving L48S and their correlation
with phenotypes . The homozygous patient for L48S had classic
PKU . Also, in combination with null mutations, L48S was associated
with severe phenotype. Our findings on the effect of other mutations
were mainly in concordance with previous European studies . However,
functional and structural effect of the S231F mutation was not
previously analyzed . Therefore, we characterized S231F PAH protein
in prokaryotic and eukaryotic expression systems . In both systems the
mutant enzyme was unstable . Its residual enzyme activity was lower
than 10% showing that S231F is a severe mutation . We have found no
GroESL chaperone effect and slightly positive effect of the BH4 on the
stabilization of the protein structure. These findings elucidated severe
phenotype of the patient with L48S/S231F genotype . In conclusion,
we showed that L48S is consistently severe PAH gene mutation in
Serbian population .
P01.067
Distribution of Xmn i alleles at the phenylalanine hydroxylase
gene in Republic of moldova
A. P. Gavriliuc 1 , S. A. Groppa 2 ;
1 National Center of Reproductive Health & Medical Genetics, Chisinau, Republic
of Moldova, 2 State University of Medicine & Pharmacology, Chisinau,
Republic of Moldova.
Phenylketonuria (PKU) is a common autosomal recessive disease of
amino acid metabolism caused by phenylalanine hydroxylase (PAH)
deficiency. The human PAH gene has been localized to 12q22-24,
comprises 13 exons spread over 90 kb of genomic DNA (Lidsky, 1984) .
The complete 2,4 kb cDNA (Kwok, 1985) can be used to detect ten different
RFLPs located within the PAH locus .
Genomic DNA was extracted and examined by standard procedures
from 68 families with classical PKU, i .e . 272 parental chromosomes .
PCR amplification of a 205 bp fragment containing the Xmn I RFLP site
was performed as described previously by Goltsov (1992) . A total 136
mutant and 140 normal chromosomes were analyzed for Xmn I polymorphic
restriction site (GAANN/NNTTC) at the PAH gene region .
Frequencies of normal alleles in population Republic of Moldova
(0,593; 0,407) are not significantly different from European populations
(0,618; 0,382) (χ 2 = 0,2; p>0,7) . The level of observed heterozygosity in
population of Moldova (0,48) is similar with European countries (0,47) .
The distribution of mutant PKU alleles (0,772; 0,228) in our study is
differs significantly from those observed in European (0,654; 0,346) (χ 2
= 4,9; p