2009 Vienna - European Society of Human Genetics

Genetic analysis, linkage ans association
found genetic variants of MMP-3 and ACE genes that might be considered
to have a protective effect.
The aim of the study was to evaluate combined effect of 5A/6A genotypes
of MMP3 and I/D genotypes of ACE genes in development of
dilatative pathology of ascending thoracic aorta (DPATA).
Material and methods. We studied 94 (68 males, 25 females) patients
with DPATA, the age ranged from 31 to 81 years (median, 64 years)
and a random sample of the population consisting of 248 males and
317 females aged 45 to 72 years (median, 60 years) all from Lithuania.
Analysis was done on DNA using conventional and real time PCR to
genotype polymorphism 5A/6A at a position -1171 of the MMP3 gene
promoter and polymorphism I/D of the ACE gene.
Results. It was tendency of higher prevalence (p=0.09) of MMP-3 promoter
5A/5A and ACE D/D genotypes in the patients with DPATA compared
to the persons from the random sample of population, 8.51 %
and 5.13%, respectively.We did not find any carrier of combined MMP-
3 promoter 6A/6A and ACE I/I genotypes in patients with DPATA. In
contrast, 7.61% of the random sample population have this combined
gene variant. The resulting p value was p=0.003.
In conclusion, the frequency of MMP-3 promoter 6A/6A and ACE I/I
genotypes might be considered as a gene combination protecting from
development of dilatative pathology of ascending thoracic aorta.
P17.46
characterization of potential splice site mutations in the LDL
receptor gene in patients with clinical diagnosis of Familial
Hypercholesterolaemia
A. C. Alves, V. Franscisco, A. M. Medeiros, L. Marques, M. Bourbon;
Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal.
Familial Hypercholesterolaemia (FH) is an inherited disorder characterized
by a high concentration of serum low-density lipoprotein (LDL)
cholesterol since birth, leading to early development of atherosclerosis
and coronary heart disease. FH is most commonly caused by mutations
in LDLR gene. About 6% are located near a slice junction or within
intron, predicted to affect the correct splicing of the mRNA.
A total of 11 potential splice site mutations, were found in the “Portuguese
FH Study”. Six alterations, five of them novel, needed further
investigation. RT-PCR of LDLR mRNA isolated from fresh blood
mononuclear cells of each patient was performed to test the effect
of the alteration on splicing. Three of these variants (c.1060+1G>A,
c.2140+1G>A, c.2547+1G>A) caused exon/exons skipping; c.190+4
insTG caused retention of two nucleotides of intron 2, and c.818-2G>A
caused retention of 10 nucleotides of intron 5. One variant could not
be tested experimentally, but it almost certainly affect splicing because
disrupts the invariant GT donor spice sites (1845+1delG). All these
alterations lead to a premature termination of the protein which means
that the protein coded by the affected allele is smaller and has no function,
most possible is degraded within the cell. To correctly estimate
the number of transcripts that are not correctly spliced, real time PCR
should be performed. This will be the next experimental step.
It is important to functional assay the effect on splicing of the alterations
found to prevent genetic misdiagnosis of FH patients and to determine
the severity of the disease.
P17.47
Familial hypercholesterolemia and premature heart disease
A. M. Gaspar 1,2 , I. M. Gaspar 3,4 , S. Martins 1 , R. Rossi 4 , R. Ferreira 4 , F. M.
Martins 4 , H. Santos 5 , I. Gomes 6 , O. Moldovan 6 , J. Henriques 7 , A. M. Silva 8 , N.
Vasconcellos 9 , A. C. Alves 10 , A. C. Medeiros 10 , M. Bourbon 10 ;
1 Metabolic Unit, Pediatric Departement, Santa Maria Hospital, Lisboa, Portugal,
2 Santa Maria Hospital, Lisboa, Portugal, 3 Medical Genetics Department,
Egas Moniz Hospital, Lisboa, Portugal, 4 Cardiologic Pediatric Department,
Santa Cruz Hospital, Lisboa, Portugal, 5 GENOMED, Lisboa, Portugal, 6 Medical
Genetics Department,, Santa Maria Hospital, Lisboa, Portugal, 7 Medicine
Department, Egas Moniz Hospital, Lisboa, Portugal, 8 Medicine Department,
Egas Moniz Hospital, Lisboa, Portugal, 9 Cardiologic Department, Egas Moniz
Hospital, Lisboa, Portugal, 10 Unidade I&D, Grupo Investigação Cardiovascular,
Instituto Nacional de Saúde, Lisboa, Portugal.
Familial Hypercholesterolemia (FH) is an autosomal dominant disorder,
usually caused by mutations on LDLR, APOB and PCSK9 genes
which is associated with premature atherosclerosis and premature
coronary heart disease (CHD).
Objective: To asses the ability to achieve clinical and molecular char-
acterization of patients and relatives with family history of premature
CHD.
Material And Methods: A clinical questionnaire of the “Portuguese FH
Study” was filled for all patients which included the characterization
of premature CHD and lipid profile. Mutations in LDLR, APOB, and
PCSK9 genes were analysed by PCR amplification and sequencing,
in a retrospective and prospective study including 80 index cases (IC)
and 110 relatives.
Results: A total of 22/190 (11.57%) patients, 14/80 (17.5%) IC and
8/110 (7.27%) relatives, had premature CHD, (15M/1F). The mean
total cholesterol (CT) was 342.39±69.46mg/dl, before treatment (BT)
and 269.28±22.09mgdl, after treatment (AT). The mean LDLC was
274.15±57.48mg/dl (BT) and 190.94±16.32mg/dl (AT). Five novel mutations
resulting in a splicing error were identified: (c.2547+1 G>A),
(c.2140 +5 G>A), (c.1359-5C>G), (c.190+4ins TG), and 1016_1017insG
(L318fsX336). These mutations were characterized functionally by
LDLR mRNA studies. The other mutations are: c.-42C>G, (EX16_18
del), c.1085delA (D341fsX348), c.530C>T (S156L) and a compound
heterozygous [c.670G>A (D203N)] + [c.2146G>A (E695K)].
Conclusion: Male patients with LDLR mutations present a more severe
phenotype since they develop premature CHD more often than
women. The early identification of FH patients can preventing the development
of premature CHD if patients receive appropriate pharmacological
treatment.
P17.48
Portuguese FH study: the genetic screening of Familial
Hypercholesterolaemia in Portugal
A. M. Medeiros, A. C. Alves, S. Silva, V. Francisco, M. Bourbon, On behalf of
the investigators of the Portuguese FH Study;
Departamento de Promoção da Saúde e Doenças Crónicas, Unidade de I&D,
Grupo de Investigação Cardiovascular, Instituto Nacional de Saúde Dr. Ricardo
Jorge, Lisboa, Portugal.
Familial Hypercholesterolaemia (FH) is an autosomal dominant disorder
with a frequency of 1/500 in most European countries and Portugal
should have about 20000 cases. FH is clinically characterized by high
levels of plasma cholesterol, leading to premature atherosclerosis and
coronary heart disease (CHD).
The aim of the Portuguese FH Study is to identify FH patients in order
to prevent the development of premature CHD.
The genetic diagnosis is based on molecular study of LDLR, APOB
and PCSK9 genes including techniques such as PCR, DHPLC, automated
sequencing and MLPA. Blood samples were collected from
343 index patients with clinical diagnosis of FH and 686 affected/unaffected
relatives. A total of 320 individuals were identified with a genetic
defect in one of these genes: 315 heterozygous, 3 compound heterozygous
and 2 true homozygous. Sixty eight different mutations were
detected in LDLR gene, 38 previously described and 30 exclusive of
Portuguese population, including 12 missense mutations, 1 nonsense,
8 splice site mutations, 7 small deletion/insertion and 3 large deletions.
APOB3500 mutation was identified in 9 individuals and one carried a
non described mutation in APOB gene. One novel mutation in PCSK9
gene was identified in three individuals.
Although the Portuguese FH Study only identified until now 1,7% of
FH cases estimated to exist in Portugal, the genetic diagnosis and
counselling of these patients should result in the establishment of appropriate
treatment and adoption of a healthier lifestyle, allowing them
to obtain a quality and life expectancy similar to a healthy person.
P17.49
the molecular diagnostics of familial hypercholesterolemia in
czech population
R. Goldmann 1 , L. Gojova 1 , P. Zapletalova 1 , L. Tichy 1 , T. Freiberger 2 , L.
Fajkusova 1 ;
1 Centre of Molecular Biology and Gene Therapy, Brno, Czech Republic, 2 Centre
for Cardiovascular Surgery and Transplantation, Brno, Czech Republic.
Familial hypercholesterolemia (FH) is an autosomal dominant disorder
caused by mutations in the gene encoding the low density lipoprotein
receptor (LDLR). The frequency of heterozygotes is 1/500.
The frequency of homozygotes or compound heterozygotes is 1/1
000 000. Mutations in the LDLR gene were determined using exon
by exon screening methods based on individual exon amplification,
DHPLC analysis and sequencing. We found 14 novel causal mutations