2009 Vienna - European Society of Human Genetics

Metabolic disorders
sian Federation.
We describe clinical data of 9 patients with mutations in the gene, coding
the mitochondrial polymerase gamma (POLG). Mutations in the
POLG gene are one of the most common causes of mitochondrial disease
in children and adults.
We investigate 3 adult patients with clinical picture of mitochondrial
disease. One patient, a 62-old woman with severe ataxia, external
ophtalmoplegia, paresthesia in legs, autoimmune thyroiditis and myopathy.
She has W748S mutation in POLG gene. Two patients had
A467T and P587L mutations consequently and showed typical symptoms
of CPEO.
We investigate six children with hepatopathy and myopathy.
Four patients from this group had Alpers syndrome: intractable seizures
with a focal component, severe neurological deterioration, hepatic
failure. Additional symptoms were vomiting, ataxia, hypotonia,
high levels of lactate, liver transaminase and high concentration of
tyrosine in urine. Mutant POLG alleles were detected in all Alpers syndrome
cases (G268A/A467T; W748S/G848S; W748S/L311P; W748S/
T855S). Symptoms of the rest two patients weren’t suitable for classical
Alpers syndrome. They both didn’t have epilepsy, but have very
severe hypoglycemia with severe hepatic failure and muscle hypotony.
We revealed only one mutant POLG allele in each case, A467T and
W748S consequently. mRNA analysis didn’t detect exon deletions in
POLG gene. The presence of the second mutant allele in the promotor
region or locus heterogeneity involving mutations in other “mitochondrial”
genes can not be excluded.
Clinical phenotypes of POLG-related disorders are characterized by
extremely variability of symptoms and this group may be more common
than previously thought.
P13.03
Evaluation of disease relevant mutations in Anderson Fabry
Disease - how to proof the medical consequences?
J. Lukas 1 , J. Frahm 1 , S. Weiss 1,2 , A. Wree 3 , R. Köhling 4 , A. Rolfs 2 ;
1 Albrecht-Kossel-Institute for Neuroregeneration, University of Rostock,
Rostock, Germany, 2 Centogene GmbH, Rostock, Germany, 3 Department of
Anatomy, University of Rostock, Rostock, Germany, 4 Department of Physiology,
University of Rostock, Rostock, Germany.
Anderson Fabry Disease (AFD) is an X-linked hereditary disorder of
sphingolipid metabolism caused by a genetic defect of the lysosomal
hydrolase α-galactosidase A (AGLA). So far, more than 300 AGLA
missense mutations have been described. However, numerous amino
acid exchanges appear to have only mild effects on enzyme activity
reduction.
Bioinformatic tools cannot reliably predict the effects of these changes
and inevitably need to be supported by enzymatic measurements.
Therefore, a rapid screening procedure for all AGLA mutations will be
invaluable for this purpose. To solve the limitations of all routine methods
we developed an in vitro model based on the overexpression of
AGLA mutants using a HEK293 cell system. Using fluorescence enzyme
activity assays we are able to determine the activity of mutant
enzymes compared to the wildtype. This procedure has shown that
the following mutations, which are described in the literature or have
been found in our screening programs for Fabry have to be interpreted
as SNPs and not clinically relevant AGLA mutations: D83N, S102L,
S126G, R220Q, R252T.
Tools for the systematic in-vitro evaluation of newly detected mutations
are also of high value for the evaluation of the consequences of new
therapeutic strategies, e.g. using pharmacological chaperones (PCs)
such as 1-Deoxygalactonojirimycin (DGJ). One of the challenges is
to find the right PC for any particular mutation because a substance
might perform well on one mutation while failing on another. The present
study includes a solution for screening newly synthesized drugs to
discover substances capable of acting like PCs.
P13.04
the molecular landscape of genetic B12 metabolism disorders
in patients from mediterranean and Latin- American origin
B. Pérez, B. Merinero, A. Rincón, A. Jorge-Finnigan, S. Brasil, F. Leal, L. R.
Desviat, M. Ugarte;
Centro de Biología Molecular. Universidad Autónoma de Madrid, Madrid, Spain.
Vitamin B 12 is the cofactor of Methionine synthase (MTR) and Methylmalonyl-CoA
Mutase (MCM) that catalyze the remethylation of homo-
cysteine and the isomerization of L-methylmalonyl-CoA, respectively.
To date defects in ten different genes coding for proteins involved in
B 12 transport, synthesis of active cofactors-MetCbl and AdoCbl- and
for the two apoenzymes MCM and MTR have been described. We
report the molecular analysis of 65 cases with isolated methylmalonic
acidemia (MMA) and 32 with methylmalonic acidemia combined
with homocystinuria (MMACH) mainly from Mediterranean and Latin-
American origin referred to our laboratory. Using cellular, enzymatic
and genetic approaches the MMA patients were classified belonging
to the cblA (n=13), cblB (n=6), cblD-variant2 (n=3) and mut (n=43)
complementation groups, and the MMACH patients belonging to the
cblC group (n=32). The mutational spectrum in the mut affected patients
includes a high frequency of missense changes (56%). Two
deep intronic changes causing intronic sequence exonization were
found and in vitro antisense therapy was investigated. In cblB affected
patients intronic and exonic nucleotide changes affecting splicing were
frequent, and two misfolding missense mutations have been functionally
analyzed. Finally, in cblA, cblC and cblD-variant2 complementation
groups a high proportion of premature stop codon changes (PTC),
including nonsense and frame-shift mutations, has been identified. In
cblC type patients the highest frequency described to date of the common
change R91fs (89%) has been found. The present work has a diagnostic
and therapeutic value in this complex metabolism addressing
our investigation towards genetic specific therapies.
P13.05
study of apoptosis in genetic B metabolism disorders (cblB
12
and cblc types)
A. Jorge-Finnigan1,2 , B. Pérez1,2 , A. Gámez2 , M. Ugarte1,2 , E. Richard1,2 ;
1Centro de Biología Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain,
2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-
ER), Madrid, Spain.
B metabolism disorders are a heterogeneous group of rare genetic
12
metabolic diseases caused by defects on cobalamin adsorption, transport
or function. The aim of this study was to analyze apoptosis in
several patients´ fibroblasts with defects on two of the genes involved
(MMAB and MMACHC) by flow cytometry, as well as to investigate
the differential gene expression in pathways that regulate this process
by PCR array. Patients´ cell lines showed a higher rate of apoptosis
compared to controls. Using the human apoptosis pathway array,
we examined the gene expression profiles exhibited by six cblC and
two cblB patients´ fibroblasts relative to four control individuals. Of
the 84 apoptosis pathway-focused genes in the array, a total of 31
genes were differentially expressed. Up-regulation was observed in
23 genes, while 8 genes appeared to be down-regulated in patients´
fibroblasts. The most highly up-regulated genes belong to BCL2 and
TNF receptor and ligand functional gene families. Multiple anti-apoptotic
genes, including BAG1, BCL2, BCL2A1, BCL2L1, NAIP, BRAF,
MCL1, TNF, CD27 and IGF1R were up-regulated; meanwhile BFAR
was down-regulated. PCR array results are currently being confirmed
by Western Blot. In addition, we have detected the up-regulation of the
intracellular modulators of apoptosis, JNK and p38 MAP kinases, in
patients´ cells by immunoblotting. In conclusion, the increased rate of
apoptosis in cblB and cblC patients´ fibroblasts might up-regulate the
anti-apoptotic molecules representing a compensatory response to a
potential disease pathogenic mechanism.
P13.06
Novel mutation in the GCH gene causing GtP-6 cyclohydrolase
deficiency in two saudi sibs and early detection by serum
neopterin
N. M. AL-HASHMI1 , M. Al-Owain2 ;
1 2 king fasial special hospital and research center, riyadh, Saudi Arabia, King
fasial special hospital and research center, Riyadh, Saudi Arabia.
GTP-6 cyclohydrolase (GTPC) deficiency is a rare autosomal recessive
disorder of the terahydrobiopterin (BH4) pathway. Only 5 patients
reported in the literature. GTPC deficiency characteristic by hyperphenylalaninemia
and low concentration of neopterins in urine and
blood and neurotransmitter defect. We report tow sibling diagnosed
with GTPC deficiency, one late and other early diagnosis. Patient 1
presented at the age of nine years with severe global development
delay and frequent twitching of body. The plasma amino acid showed
a phenylalanine of 357 umol/l.Which was not consistent with classical