12th Congress of the European Hematology ... - Haematologica

wave length of light of reflected ray, chemical composition of sample,
thickness of sample, refractive index of sample, refractive index of substrate,
chemical composition of substrate. The setup for color reflected
interference microscopy was centered around ordinary optical microscope
(Carl Zeiss, Germany) equipped for digital photo camera (Sony)
and substrate which serve as object-plate for sample and as source of
coherent light for scattering on morphological structures of sample. Light
from a 100 watt xenon source was directed on to the specimen. Microscopic
images were obtained with Zeiss lens and digital camera and
recorded on a personal computer using commercially available software.
Results. To demonstrate the potential usefulness of this method, we provide
qualitative data describing color image of healthy and pathological
damaged cells for alive and dry blood smears. Comparison Figure 1A and
Figure 1B for same samples but obtained by conventional bright-field
microscopy and by using new method correspondently showed distinguishing
in color not only separate red blood cells but distinguishing difference
parts in area separate erythrocytes too. Usually for healthily individuals
a albuminous aureole around the erythrocytes are mainly whiteyellow
but for cancer cells (core rectal cancer) the aureole color is quite
different and reflects significant changes in chemical composition of
both internal, and external contents of erythrocytes (Figure 1D, 1E). Easy
detection of organic shells around blood cells in our case is evident.
Operations by fixing, smear coloring, prolonged processing, the availability
for phase-contrast or interference microscope, special illuminators,
radiating the exciting short-wave light beams are not required. Interferometric
coloring of blood elements occurs on a surface of specially selected
substrate. Corresponding colored images of blood elements are
formed due to interference phenomena occurring under interaction of
light beams reflected from front and back surfaces of blood elements,
smeared on a substrate. As it is seen from the given micro photos, the
character of the colored image is the same, as though they were investigated
with phase-contrast or interference universal microscopes.
References
1. Kozlovskaya L.V., Nikolaev A.Yu. Textbook on Clinical Laboratory
Methods of Investigations. Moscow. Medicine 1984. 288p.
2. Zernike F. Phase contrast: A new method for the observation of transparent
objects. Physica 1942;9:686-93.
3. Nomarski G. Microinterferometric differential a on des palarisees. J
Phys.Radium 1955;16:S9.
0778
ANALYSIS OF SLC40A1 (FERROPORTIN 1) GENE AT MRNA LEVEL REVEALS RAPIDLY THE
CAUSATIVE MUTATIONS IN PATIENTS WITH HEREDITARY HEMOCHROMATOSIS TYPE IV
M. Speletas, 1 A. Kioumi,2 G. Loules, 1 F. Kalala, 1 E. Katodritou, 2
I. Tsitouridis, 2 P. Hytiroglou, 3 J. Christakis, 3 I. Korantzis, 2 A. Germenis1 1 University of Thessaly, LARISSA; 2 Papageorgiou Hospital, THESSALONIKI;
3 Aristotle University of Thessaloniki, THESSALONIKI, Greece
Background. Mutations in the SLC40A1 (ferroportin 1) gene result in a
dominant genetic disorder [ferroportin disease; hereditary hemochromatosis
type (HH) IV], characterized by iron overload. In all previous
studies, the mutational analysis of SLC40A1 gene has been performed
at genomic DNA level by PCR amplification and direct sequencing of all
coding regions and flanking intron-exon boundaries (usually in 9 PCR
reactions). The aim of this study was to analyze the SLC40A1 gene at
the mRNA level, for the rapid detection of ferroportin 1 gene alterations.
Methods. Two female patients displayed hyperferritinemia, normal transferrin
saturation and iron accumulation predominantly in macrophages
and Kupffer cells (typical ferroportin disease phenotype). mRNA was
extracted from PBMCs by standard protocol. Afterwards, the entire coding
sequence of the SLC40A1 gene was amplified in only two RT-PCR
reactions, following by direct sequencing or/and NIRCA (non-isotopic
RNase cleavage assay). Results. RT-PCR-sequencing analysis showed that
one patient displayed the previously described alteration V162? and the
other one the novel mutation R178G. NIRCA analysis demonstrated the
results of sequencing analysis in both cases, confirming that it could be
used as a first screening (but not necessary) step for the detection of
SLC40A1 gene alterations. Conclusions. This protocol turned out to be
rapid, sensitive and reliable, facilitating the detection of SLC40A1 gene
mutations in patients with hereditary hemochromatosis type IV. The
broad application of this procedure may facilitate the rapid molecular
analysis of SLC40A1 gene contributing to the understanding of the Fpn
disease pathogenesis.
12 th Congress of the European Hematology Association
0779
MOLECULAR CHARACTERIZATION OF A NEW LONG DELETION IN THE FERROCHELATASE
GENE
E. Di Pierro, 1 V. Brancaleoni, 1 V. Besana, 1 S. Ausenda, 1 S. Drury, 2
M.D. Cappellini1 1 Foundation IRCCS Maggiore Hospital-UniMi, MILAN, Italy; 2 Medical Genetics,
Children's Hospital, MONTREAL, Canada
Background. Erythropoietic protoporphyria (EPP, MIM 177000) is an
autosomal dominant disease with incomplete penetrance, due to
reduced activity of ferrochelatase (FECH; EC 4.99.1.1). The enzyme is
located in the inner mitochondrial membrane and catalyzes the chelation
of ferrous iron into protoporphyrin IX, the final step in the heme
biosynthetic pathway. Clinical manifestations have a childhood onset
and include skin photosensitivity and mild anaemia. The human ferrochelatase
gene (FECH) maps to chromosome 18q21.3; it spans 45kb
with a total of 11 exons encoding for a precusor of 423 amino acid
residues, the first 62 of which are the putative mitochondrial leader
sequence. So far molecular analysis of FECH gene has allowed the identification
of more than 100 different mutations responsible for EPP. Phenotypic
expression of EPP requires coinheritance of a null FECH allele
and a wild-type low expressed allele; there are evidences suggesting that
an entire haplotype (-251G, IVS1-23T, IVS3-48C; GTC haplotype) is
involved in reducing the allele expression. Aims. We analysed a Canadian
family of Italian origin in which the proband showed clinical signs of
EPP but no mutations were found in the promoter and in the entire coding
region. Moreover, the proband carried the GTC haplotype and the -
251G polymorphism in apparently homozigosity. Family studies established
absence of mendelian segregation for the -251G polymorphism,
suggesting an emizigosity in this region and a possible deletion. The aim
of this study was to identify the size of the putative deletion. Methods.
The promoter, the entire coding region and the intron-exon boundaries
of FECH gene have been amplified by PCR and submitted to direct
sequencing. In order to identify an heterozygous region, we extended the
SNPs analysis upstream the FECH gene and we performed XL-PCR
analysis to establish the size of the deletion. Results. Two heterozygous
polymorphisms were found on asparaginyl-tRNA synthetase (NARS)
gene. Thus we conducted an XL-PCR using primers situated in the NARS
gene and in the intron 2 of FECH gene respectively. The XL-PCR showed
a wild type fragment of 14569bp and a shorter fragment of about 4000
bp. Sequence analysis on the isolated abnormal fragment showed a
10377bp deletion. The deletion includes an intergenic region of about
6500bp, the promoter, the exon 1 and part of intron 1 of the FECH gene.
The family study identified also two asymptomatic carriers of the same
mutation. Conclusions. This deletion causes the loss of the entire promoter
which probably prevents the expression of the mutated allele.
Nevertheless this allele could code for an mRNA lacking the first 22 aa
of the leader sequence, causing the retention of the protein in the cytoplasm
and thus the blockage of its translocation to the inner mitochondrial
membrane. Quantitative RNA analysis is under investigation to
confirm these hypothesis. The deletion is probably caused by unequal
intragenic recombination between two Alu sequences which have been
found close to the breakpoints. The presence of several Alu in the FECH
gene suggests the high probability of deletions as a cause of EPP.
0780
MOLECULAR HETEROGENEITY OF PORPHYRIA CUTANEA TARDA IN ITALY:
IDENTIFICATION OF THREE NOVEL MUTATIONS IN THE UROPORPHYRINOGEN
DECARBOXYLASE GENE
E. Di Pierro, V. Brancaleoni, S. Ausenda, V. Besana, D. Tavazzi,
M.D. Cappellini
Foundation IRCCS Maggiore Hospital-UniMi, MILAN, Italy
Background. Porphyria cutanea tarda (PCT, MIM 176100) is a human
metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen
decarboxylase (URO-D, E.C. 4.1.1.37) activity, the fifth
enzyme of the heme biosynthetic pathway. A classification of inherited
and non-inherited forms is based on the enzyme activity levels in red
blood cells (RBC). Among the diagnostic criteria, the most powerful is
the URO-D deficiency and the presence of a plasma fluorescent peak at
620 nm. The main clinical findings observed in PCT are skin lesions on
light-exposed areas of the body, skin fragility, vesicles and bullae formation
after sun exposure. Clinical manifestations of PCT are often precipitated
by triggering factors such as alcohol, drug abuse, estrogens, virus
infections, hepatotoxic chemicals and hepatic siderosis. Nowadays more
than 70 molecular abnormalities have been so far identified in the URO-
haematologica/the hematology journal | 2007; 92(s1) | 291