2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
I statues. We also show that the HS-111 (-21 A>G) variation correlates
with increased fetal hemoglobin production in β-thalassemia intermedia
and major patients. In contrast, the 3`HS-1 (+179 C>T)) mutation is
not statistically significant. We conclude that the -588 A>G and HS-111
(-21 A>G) variations are useful genetic determinants for differentiation
of β-thalassemia major and intermedia patients. However this nucleotide
change alone may not be sufficient to raise the level of HbF, other
unknown factors may play a role in HbF production.
P12.003
Further analysis with multiplex Ligation dependent Probe
Amplification (MLPA) of the ABCA gene in spanish patients
with retinal dystrophies
J. Aguirre-Lamban1,2 , R. Riveiro-Alvarez1,2 , D. Cantalapiedra1,2 , M. Garcia-
Hoyos1,2 , A. Avila-Fernandez1,2 , C. Villaverde-Montero1,2 , M. Trujillo-Tiebas1,2 , C.
Ramos1,2 , C. Ayuso1,2 ;
1 2 Fundacion Jimenez Diaz, Madrid, Spain, Centro de Investigacion en Red de
Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain.
Introduction: ABCA4 mutations have been associated with autosomal
recessive Stargardt disease (arSTGD). A few cases with autosomal recessive
cone-rod dystrophy (arCRD) and autosomal recessive retinitis
pigmentosa (arRP) have also been found to have ABCA4 mutations.
Comparative genetic analyses of ABCA4 variation and diagnostics
have been complicated by substantial allelic heterogeneity. The objective
of this study was to determine whether deletions and duplications
in the ABCA4 gene are a frequent cause of retinal dystrophies among
Spanish patients.
Subjects And Methods: We analyzed a total of 55 unrelated families.
Mutation analysis was performed in 40 arSTGD families, 6 arCRD
families and 9 arRP families. DNA samples from patients were previously
studied with the ABCR400 genotyping microarray. Patients with
either none or only one mutant allele were analysed with multiplex
ligation dependent probe amplification (MLPA). Sequencing was employed
for the study of 50 control samples.
Results: MLPA allowed us to find one novel mutation in heterozygosis
(p.Gln841Lys) in exon 16 of the ABCA4 gene. This variant was located
in the target site of the probe. Thus, a lower peak was shown in
the pattern of peaks of the MLPA. The p.Gln841Lys mutation was not
found in 100 control chromosomes. Neither deletions nor duplications
were found.
Conclusions: MLPA was mainly designed to detect deletions and duplications
of one or more exons of the ABCA4 gene. However, these
types of mutations are not a frequent cause of these retinopathies.
Nevertheless, this technique enabled us to additionally detect point
mutations.
P12.004
Progressive Familial intrahepatic cholestasis type 3: ABcB4
mutations in familial cases
D. Degiorgio 1 , C. Colombo 2,3 , M. Castagni 1 , M. Seia 1 , L. Costantino 1 , L. Porcaro
1 , V. Paracchini 1 , D. A. Coviello 1 ;
1 Laboratorio di Genetica Medica, Fondazione IRCCS, Ospedale Maggiore
Policlinico, Mangiagalli e Regina Elena, Milan, Italy, 2 Centro Fibrosi Cistica,
Fondazione IRCCS, Ospedale Maggiore Policlinico, Mangiagalli e Regina
Elena, Milan, Italy, 3 Dipartimento di Pediatria, Università degli Studi di Milano,
Milan, Italy.
The ABCB4 protein translocates phosphatidylcholine from the inner to
the outer leaflet of the canalicular membrane of the hepatocyte (“floppase”
activity). Severe ABCB4 deficiency causes Progressive Familial
Intrahepatic Cholestasis type 3 (PFIC-3) characterized by early onset
of persistent cholestasis that progresses to cirrhosis and, frequently,
to end-stage liver disease before adulthood. We enrolled 132 children
with PFIC-3 phenotype and 100 healthy subjects and sequenced all
the 27 coding exons of ABCB4 gene. We observed 31 distinct disease
associated-mutations in 27 patients from 22 families including 4 families
with more than 1 affected sibling. In family A, two brothers carried
two null alleles that caused death and liver transplantation at the age
of 5 and six years, respectively. In family B two siblings, aged 12 and
2 respectively, carried two missense mutations (compound heterozygous)
associated to compensated cirrhosis on both, and with portal
hypertension in the oldest. In family C two siblings, a 6-year-old girl
and a 4-year-old boy, carried three missense mutations with double
paternal mutant allele associated with compensated cirrhosis in both
brothers and episodes of clinical cholestasis in the oldest. In family
D, three siblings carried three missense mutations with double maternal
mutant allele; liver transplantation was required in the 17 year
old boy, compensated cirrhosis was documented in the 12 year old
boy whereas any relevant clinical symptom was found in the youngest.
ABCB4 deficiency associated with two mutated alleles and severe
liver failure in families with affected PFIC-3 children requires a careful
genetic counselling.
P12.005
Novel mutations in the ABcR gene associated with stargardt
maculopathy in the italian patients
I. Passerini1 , A. Sodi2 , A. Mariottini1 , S. Palchetti1 , C. Giuliani1 , U. Menchini2 , F.
Torricelli1 ;
1 2 AOU Careggi-SOD Diagnostica Genetica, Florence, Italy, AOU Careggi-II
Clinica Oculistica, Florence, Italy.
Stargardt disease (STGD) is a progressive juvenile-to-young adultonset
macular degeneration inherited as an autosomal recessive trait
(arSTGD); mutations in the ABCR (photoreceptor-specific ATP-binding
cassette (ABC) transporter) gene are responsible for arSTGD. In this
study we determined the mutation spectrum in the ABCR gene in a
group of Italian patients with arSTGD.
93 families from central Italy, some members of which were affected by
autosomal recessive Stargardt disease, were examined.
In all these patients we reported some mutations of ABCR gene. 99
mutations were identified: 61 missense mutations (P68L, I73T, N96K,
I156V, G172S, H193P, R212C, N415K, L541P, E616K, R653C, G690V,
V767D, W821R, M840R, G863A, T897I, V931M, N965S, T970P,
T977P, G978D, F1015I, T1019M, A1038V, R1055W, G1078E, E1087K,
T1089I, R1098C, R1108C, R1108H, L1201R, D1204N, P1380L,
V1433I, L1473M, P1484S, T1526M, L1580S, A1598D, S1696M,
Y1754C, A1762D, A1794D, N1805D, S1806N, H1838D, H1838N,
R1843W, G1961E, L1970F, G1977S, L2027F, V2050L, E2096K,
L2140Q, K2172R, L2221P, R2269Q, Q2272K); 12 nonsense mutations
(Q21X, R572X, W700X, E1087X, S1099X, C1177X, Q1332X,
W1408X, W1461X, W1479X, R2030X, Q2220X); 12 splicing mutation
(V256splice, IVS6-1G>T, IVS9+1G>C, IVS13+15G>A, Q1376splice,
IVS28+5G>A, IVS32+1G>A, IVS35+2T>C, IVS40+5G>A, IVS42-
2delA, IVS42+4delG, IVS45+1G>C); 8 small deletions (811delGAGA-
TG, 4733delCGTTT, 5109delG, 5917delG, 5961delGGAC, 6535delT,
6750delA, 6758delA); 5 small insertion (250insCAA, 324-327insT,
3584insGT, 6548insTGAA, 6464ins8bp); and one gross insertion
(4021ins24bp). G1961E was the most frequent in our series. 41 mutations
had not been previously described and were not detected in 150
unaffected control individuals..
These data confirm the extensive allelic heterogeneity of the ABCR
gene, in agreement with previous observations in patients with Stargardt
disease from Italy.
P12.006
identifying new AiRE interacting protein.
A. Meloni 1 , D. Corda 2 , F. Incani 2 , E. Fiorillo 2 , D. Carta 2 , A. Cao 1 , M. C. Rosatelli
2 ;
1 Istituto di Neurogenetica e Neurofarmacologia, Consiglio Nazionale delle
Ricerche, Cagliari, Italy, 2 Dipartimento di Scienze Biomediche e Biotecnologie,
Università degli Studi di Cagliari, Cagliari, Italy.
Autoimmune polyglandular syndrome type 1 (APS1), is a rare, monogenic
autoimmune disease caused by mutations in the Autoimmune
Regulator (AIRE) gene. The clinical phenotype of APECED patients
reveals a triad of main manifestations: adrenocortical failure, hypoparathyroidism
and chronic mucocutaneous candidiasis. The AIRE
protein contains several functional domains which are suggestive of
a role as a transcriptional regulator: four LXXLL motifs, one SAND domain,
one HSR domain and two PHD fingers. Herein we describe the
functional studies performed to identify new AIRE interacting proteins.
We screened a human thymus cDNA library through the yeast two
hybrid technique and we identified seven different clones encoding the
same protein. The functional domains involved in the interaction with
AIRE protein have been mapped using deletion mutants. The physical
interaction was further confirmed with several in vivo and in vitro
approaches in fact we proved the interaction in mammalian cells by
co-IP and confocal analysis and then through GST-pull down assays.
Chasing AIRE interacting proteins allowed us to discovery a new pro-
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