2008 Barcelona - European Society of Human Genetics

Clinical genetics
agarose gel . The results showed that two families are carriers for the
Asian-Indian inversion and deletion . Genotype-phenotype correlation
was performed the same as globin gene server database .
P01.006
Genotyping of α-globin genes in Iranian α-thalssemia carriers
E. Shafieiyeh, M. Karimipour, Z. Kaini Moghaddam, A. Kordafshari, S. Zeinali;
Institute Pasteur, Tehran, Islamic Republic of Iran.
Introduction:
Alpha thalassemia is more often caused by deletions involving one or
both of the α-globin genes.
A small number of point mutations, usually within the α2, have been
characterized . The aim of
This study was molecular analysis of α-globin genes in some Iranian
people with low MCV, MCH and normal HbA2 and HbF .
Materials and Methods:
After obtaining informed consent, DNA was extracted from blood samples
of 100individuals referred from Primary Health Care (PHC) centers
by salting out method. Multiplex Gap-PCR for common α-globin
gene deletions was performed . Then for individuals who did not have
known deletions, α2 and α1-globin genes were sequenced by chain
termination method .
The sequences were aligned against Z84721 accession number in
GenBank and results were compared with globin gene server database
.
Results:
Among 100 individuals, 47 individuals have had deletions in α-globin
gene including:
-α3 .7 (27), --Med (8), -α4 .2 (7), -α20 .5 (5) .
of the 53 remaining individuals who did not have known deletions in αglobin
genes,23 samples had different mutations; including: PolyA (8), 4
PolyA & 5nt (5), C .S(1),hemoglobin Adana,Cd28 . The most common
6
deletions and point mutation were -α3 .7 and PolyA, respectively .
Conclusion:Non deletion mutations can interact with each other or
α0 -thalassemia deletions to produce severe forms of HbH disease or
even Hb hydrops fetalis.Thus screening for α-thalassemia should be
considered during genetics counseling of high risk couples of thalassemia
for prenatal diagnosis. In addition, α-thalassemia may alter the
hematologic parameter in β-thalassemia carriers.
P01.007
A new polymorphism causes different restriction pattern by β-
RsaI in β-globin cluster: application in PND
A. Valaei, F. Bayat, M. Taghavi, M. Karimipoor, S. Zeinali;
Pasteur Institute of Iran, Tehran, Islamic Republic of Iran.
The β-globin cluster is located at chromosome 11 and contains five
functional genes .There are at least nine RFLP markers distributed all
over the cluster .These markers are used routinely for carrier testing,
prenatal diagnosis and haplotype analysis .
Working on the β-globin cluster haplotypes of some Iranian β-thalassemia
carriers and their families, we observed a different pattern of
digestion by RsaI restriction enzyme in many people .The aim of this
study was to find out the cause of this pattern.
This study was performed on carriers of β-thalassemia and normal
controls . After obtaining informed consent, DNA was extracted from
peripheral blood.ARMS-PCR method was exploited for finding the
common mutations in β-globin gene.PCR-RFLP was performed on
β-RsaI polymorphic site. The primers and PCR conditions are from
Weatheral & Cleggs . DNA sequencing was performed on PCR products
of β-RsaI marker by the same primers.
Some of the carriers of β-thalassemia and their parents had a different
digestion pattern in β-RsaI polymorphic site. In the carrier people different
mutations were found(IVSII-1, IVSI-110, IVSI-6, -88) .When polymorphic
restriction site of RsaI is absent in the sample, after digestion,
the 1200 bp PCR product is cut to 694, 411 and 95 bp bands due to
two constant restriction sites . In our cases the constant restriction site
at position 411 has been changed(GTAC to GCAG)in heterozygous
form . Hence, the restriction pattern by this enzyme creates 694 and
506 bp bands. This polymorphism is not associated to a specific mutation
in β-globin gene and also was found in normal control people.
P01.008
Co-inheritance of Hemoglobin D and β-Thalassemia Trait in three
iranian families
M. Taghavi, M. Karimipoor, M. Jafarinejad, L. katouzian, A. Valaei, A. Amirian,
F. Bayat, A. Kordafshari, N. Saeedi, S. Zeinali;
Pasteur Inistitute of Iran, Tehran, Islamic Republic of Iran.
β-thalassemia is the most common genetic disorder in Iran, occurring
more frequently in Northern and Southern areas . IVSII-I and IVSI-5
are the most common mutations reported in the country . HbD, a hemoglobin
variant occurs mainly in north-west India, Pakistan and Iran
and differs structurally from normal HbA at 121 positions on β chain.
Co-inheritance of HbD and thalassemia minor is not common and may
alter the Hb electrophoresis pattern . Here we report three cases with
combination of β-thalassemia and Hb D. None of them had symptoms
of profound anemia and hematological indices were similar to the βthalassemia
heterozygote . Hb variant level in carriers was increased
and no HbA was detected electrophoretically . After obtaining informed
consent, the blood samples were collected in tubes containing EDTA .
Genomic DNA was extracted using the salting out method . The mutation
in β-globin gene was revealed by ARMS-PCR technique and
confirmed by DNA sequencing. The region containing exon 3 was amplified
for HbD and the PCR product of this amplicon was digested
by EcoRI . The electrophoresis pattern suggested that all cases were
homozygote for HbD. But, molecular analysis confirmed the presence
of Cd 121 GAA>CAA in heterozygous form in combination with IVS
II-I and IVS I-5 . Hematological family study showed the mutations
and HbD are in trans position . These mutations produce an unstable
mRNA without any product and almost all of the globin output is from
the chromosome carries HbD .
P01.009
Analysis of haplotypes associated with iVsi nt 130 of beta-globin
gene reveals intriguing results
M. Feizpour 1 , P. Fouladi 1 , S. Foroughi 1 , F. Rahiminejad 1 , F. S. Hashemi 1 , R.
Vahidi 1 , S. Zeinali 2 ;
1 Kawsar Biotech Research Center, Tehran, Islamic Republic of Iran, 2 Pasteur
Institute and Kawsar Biotech Research Center, Tehran, Islamic Republic of
Iran.
Prevention of thalassemia is a national program . Premarital screening
and prenatal diagnosis (PND) is in effect . For performing PND, we
usually use direct mutation detection techniques like ARMS PCR . We
routinely screen for more than 42 mutations identified in our center
so far . If now mutation is detected we resort to direct DNA sequencing
. We also use beta-globin gene linked RFLPs or SNPs for PND to
increase the accuracy of PND . Sometimes haplotype analysis can be
very helpful as well .
We have so far analyzed more that 6394 chromosomes from carriers
of beta-thalassemia and in 16 cases the mutation was IVSI nt 130 .
This mutation is regarded as rare mutation in Iranian population . The
families were mostly from two distinct geographical areas, namely
Aghghola in Golestan province, west of Caspian Sea in the north of
Iran (6 cases or 37%) and Meshkin shahr in Ardabil province, east
of Caspian Sea, in north of Iran (5 cases or 31%) . The other 4 cases
were from Khozestan in South West and Gilan in North of Iran equally .
Since populations in these two regions do not have much in common
we decided to see if they share the same haplotype . We tested Hind
III ψβ, AvaII β, and HinfI β RFLP sites for this purpose. All cases from
Aghghola were + - + and all cases from Meshkin Shahr were - - + for
these sites .
This shows that there were two different founder effects for this mutation
.
P01.010
Non-Invasive prenatal diagnosis of β-thalassaemia by SNP
analysis using PNAs and Arrayed Primer Extension(APEX)
T. Papasavva 1 , A. Kyrri 2 , L. Cremonesi 3 , S. Galbiatti 3 , M. Kleanthous 1 ;
1 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2 Thalassaemia
Center, Archibishop Makarios III Hospital, Nicosia, Cyprus, 3 Fondazione
Centro San Raffaele del Monte Tabor, Milan, Italy.
The recent discovery of relatively abundant quantities of cell free fetal
DNA in maternal plasma and serum has opened up new possibilities
for the non-invasive prenatal diagnosis. β-thalassaemia is one of the
most common autosomal recessive single-gene disorders in Cyprus .