2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
P12.103
Novel RDH12 and RPE65 gene variants associated congenital
Amaurosis with Leber
F. Torricelli1 , S. Palchetti1 , I. Passerini1 , A. Sodi2 , C. De Sanzo1 , F. Girolami1 ,
U. Menchini2 ;
1 2 AOU Careggi-SOD Diagnostica Genetica, Florence, Italy, AOU Careggi-II
Clinica Oculistica, Florence, Italy.
Leber Congenital Amaurosis (LCA) is a severe retinal dystrophy involving
both cone and rod systems and causing severe visual impairment.
LCA is inherited in an autosomal recessive manner. Actually 11 genes
has been identified to be involved in the pathogenesis of the disease:
AIPL1, CRB1, CRX, LRAT, GUCY2D, IMPDH1, TULP1,RDH12,
RPE65, RPGRIP1, CEP290.
Even if these genes code for proteins playing different biological rate,
there is not a wide-range of phenotypes related to different mutations,
and the clinical features related to different mutations in different genes
seems to be similar.
In this study 3 patients of 3 different families were examined for RDH12,
RPE65 e GUCY2D genes, by direct sequencing.
The first patient showed one novel RDH12 missense variant
(p.Val233Glu) in homozygous state, and one previously described
missense mutation(p.Pro701Ser) in GUCY2D gene, in heterozygous
state.
The second patient showed one novel RDH12 stop variant
(p.Trp304Stop), in homozygous state. The same mutation was identified
in both clinically healthy heterozygote parents of this patient.
The third patient showed two RPE65 mutations in heterozyuos state:
the novel variant c.440_441delCA, and the described mutation
p.Arg91Trp.
Large-scale molecular screening of LCA-associated genes will probably
permit a better understanding of the physiopathological consequences
of the different gene mutations with more reliable prognostic
evaluations in clinical practice.
P12.104
A new splicing mutation in HPRt1 gene in a Romany boy - a
genotype-phenotype reference
J. Behunova 1 , J. Ferenczova 1 , L. Stolnaja 2 , H. Vlaskova 2 , L. Dvorakova 2 , L.
Podracka 1 ;
1 Safarik University Children Hospital, I. Department of Pediatrics, Kosice, Slovakia,
2 Charles University in Prague, 1st Medical Faculty, Institute of Inherited
Metabolic Disorders of 1st Faculty of Medicine and General Teaching Hospital,
Prague, Czech Republic.
Lesch-Nyhan syndrome is an X-linked disorder of purine methabolism
caused by Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
deficiency. Mild forms are termed X-linked hyperuricaemia or Kelly-
Segmiller syndrome, clinically presenting with gout, eventually also
mild neurological symptoms. Severe HPRT deficiency additionally
leads to nephrolithiasis with chronic renal failure and serious neurologic
impairment - psychomotor retardation, hypotonia, automutilations.
The Romany patient described here clinically presented with acute renal
failure already in newborn age. His present status (age 3 years)
represents severe delay - no sitting, no speach, hypotonia, dyskinesis.
Self-biting started in his 2nd year of life. Renal functions are reduced,
ultrasound shows kidney calcifications.
Analyzing a HPRT1 gene of the patient, we have identified a novel
splicing mutation c.27+2T>C in intron 1 (IVS 1+2T>C). The influence
of mutation´ impact to mRNA splicing was evaluated by cDNA analysis.
Sequencing of cDNA containing exons 1,2 and a part of 3, prooved
defective splicing of mRNA. Mutation c.27+2T>C abolishes the natural
donor splice site and an alternative splice site within intron 1 is used
(r.27_28ins49). The protein translated from the mutated RNA is predicted
to contain only 26 amino acid residues. However, according to
our results, the majority of mutated mRNA undergoes nonsense-mediated
mRNA decay and the defective protein is not synthesized.
The severe mutation described here occured de novo in the patient
and led to full-blown Lesch-Nyhan syndrome. Our results in accordance
with published data point to a good genotype-phenotype correlation
in patients with HPRT deficiency.
Grants´ support: VZ MSM CR 0021620806, VZ MZ CR 64165
P12.105
c-terminal deletions of y + LAt-1 do not affect the dimerization of
y + LAt-1/4F2hc transporter but can cause a targeting defect.
M. Toivonen 1,2 , K. Huoponen 1 , O. Simell 3 , J. Mykkänen 3 ;
1 Department of Medical Biochemistry and Genetics, Institute of Biomedicine,
University of Turku, Turku, Finland, 2 Turku Graduate School for Biomedical Sciences
(TuBS), Turku, Finland, 3 Department of Paediatrics, University of Turku,
Turku, Finland.
Lysinuric protein intolerance (LPI) is an autosomal recessive disorder
of cationic amino acid transport at the basolateral plasma membrane
caused by mutations in the SLC7A7 gene encoding y + LAT-1 protein.
The active amino acid transporter consists of the light subunit y + LAT-1,
which determines the substrate specificity as the heavy subunit 4F2hc
guides the complex to the membrane. 4F2hc also regulates other cellular
functions including cell activation, proliferation, survival and apoptosis.
We reported previously that y + LAT-1 protein with LPI missense mutation
is carried to the plasma membrane, while frameshift mutants are
cytoplasmic. Also, truncated y + LAT-1s lacking part of C-terminal tail are
correctly localized while larger deletions remain subcellular. However,
the y + LAT-1-4F2hc interaction occurs regardless of the targeting mutations.
In the current study, we use C-terminal deletions of y + LAT-1 to
study their dimerization using flow cytometry FRET and the effects of
their expression on the cells.
Our results indicate that the interaction of y + LAT-1 and 4F2hc within
the cell is not disrupted by any of the deleted y + LAT-1 proteins. The
localization of the small deletions is similar to the wild type, whereas
proteins lacking more than 60 amino acids remain cytoplasmic. Transfection
of C-terminally deleted y + LAT-1s results in reduction of y + LAT-
1-CFP expressing cells compared to transfection of wild-type y + LAT-1.
In addition, expression of truncated y + LAT-1 affects cellular viability,
which may be due to scavenging of 4F2hc by the mistargeted cytoplasmic
y + LAT-1, leading to shortage of 4F2hc in the plasma membrane
and reduced cell proliferation compared to the cells expressing
normally targeted 4F2hc.
P12.106
Gene expression profiling of haematological and immunological
deficiencies in lysinuric protein intolerance (LPI) patients
J. Salmi 1 , M. Tringham 1 , L. Tanner 2 , K. Huoponen 1 , K. Näntö-Salonen 2 , H. Niinikoski
2 , O. Simell 2 , J. Mykkänen 2 ;
1 Department of Medical Biochemistry and Genetics, University of Turku, Turku,
Finland, 2 Department of Paediatrics, University of Turku, Turku, Finland.
Lysinuric protein intolerance (LPI; MIM222700) is a rare autosomal recessive
disorder caused by a defect of cationic amino acid transport
in the small intestine and kidney tubules. All Finnish patients share
the same homozygous mutation c.1181-2A>T (c.859-2A>T) in the
SLC7A7 gene. Altogether 51 mutations have been found worldwide
but the Finnish founder mutation has not been detected in any other
population. The main symptoms of LPI comprise protein aversion after
weaning, failure to thrive, muscle hypothonia, osteoporosis and hepatosplenomegaly.
However, some findings vary markedly even in the
same family, and may include severe renal and pulmonary complications,
such as end-stage renal disease and alveolar proteinosis. Some
patients suffer from normochromic anaemia with poikilocytosis and anisocytosis
and immunological problems with leukopenia and deficiencies
in T- and B-cell functions.
We have used microarray technology and quantitative real-time PCR
to find out which other genes than SLC7A7 may affect the phenotype
of the patients. The gene expression profiles were obtained from the
peripheral whole-blood cells. We found systematic up-regulation of
genes encoding proteins participating in erythropoiesis, heme synthesis,
erythrocyte membrane structure, transport, enzymatic functions
and blood group antigens. Down-regulated genes encoded e.g. interleukins,
interleukin receptors, chemokines, regulators of complement
activity and histocompatibility complex proteins. These differentially
expressed genes may, indeed, light the background of haematological
and immunological problems observed in LPI patients.