European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Molecular and biochemical basis of disease
P0866. Bisulfite based methylation analysis of the 6 th CTCF
target site within the IGF2/H19 imprinting centre region 1 (ICR1)
has a higher sensitivity compared to Southernblot analysis of
the 3 rd CTCF target site
M. Zeschnigk 1 , B. Albrecht 1 , K. Buiting 1 , T. Eggermann 2 , J. Gromoll 3 , S. Kaya-
Westerloh 1 , S. Seeland 1 , B. Horsthemke 1 ;
1 Institut für Humangenetik, Universitätsklinikum Essen, Essen, Germany, 2 Institut
für Humangenetik, Universitätsklinikum Aachen, Aachen, Germany, 3 Institut
für Reproduktionsmedizin, Universität Münster, Münster, Germany.
Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous
syndrome characterized by severe pre- and postnatal growth
retardation, body asymmetry and a typical facial phenotype including
triangular face and relative macrocephaly. Apart from rare families,
most patients are sporadic. Approximately 10 % of patients have maternal
uniparental disomy of chromosome 7. In another 30 % of patients,
the differentially methylated IGF2/H19 imprinting centre region
(ICR) at 11p15 is hypomethylated. This region contains seven CTCF
target sites (CTSs). In previous studies methylation of a HpaII restriction
site close to CTS3 was analysed by Southern-blotting (Gicquel et
al., 2005). Using bisulfite treatment and a real time PCR based methylation
assay (QAMA), we determined the methylation at CTS6 in 15
patients who showed a normal CTS3 methylation by Southern analysis.
We observed CTS6 hypomethylation in 6 patients. The degree of
methylation ranged from 4 - 16 %. By Multiplex Ligation-dependent
Probe Amplification (MLPA) we extended the methylation analysis to
four additional ICR loci located between 300bp upstream and 2150bp
downstream of CTS6 and found similar degrees of hypomethylation in
all of the six patients. As transmission of SRS has been observed, we
analysed the degree of methylation in spermatozoa from one patient
and found complete methylation of CTS6, which is consistent with a
normal methylation pattern.
P0867. Heterogeneity of NSD1 alterations in 116 patients with
Sotos syndrome
P. Saugier-Veber 1 , C. Bonnet 1 , A. Afenjar 2,3 , V. Drouin-Garraud 1 , C. Coubes 4 ,
S. Ferenbach 1 , M. Holder-Espinasse 5 , J. Roume 6 , V. Malan 7 , N. Jeanne 7 , C.
Baumann 8 , D. Héron 9 , A. David 10 , M. Gérard 8 , D. Bonneau 11 , D. Lacombe 12 , V.
Cormier-Daire 13 , T. Billette de Villemeur 2 , T. Frebourg 1 , L. Burglen 7 ;
1 Department of Genetics, Rouen University Hospital and Inserm U614, Institute
for Biomedical Research, Rouen, France, 2 Department of Neuropediatrics,
Trousseau University Hospital, AP-HP, Paris, France, 3 Centre de référence des
anomalies du développement embryonnaire, Trousseau University Hospital,
AP-HP, Paris, France, 4 Department of Medical Genetics, Montpellier University
Hospital, Montpellier, France, 5 Department of Medical Genetics, Lille University
Hospital, Lille, France, 6 Department of Medical Genetics, Poissy-Saint Germain
en Laye Hospital, Poissy, France, 7 Department of Genetics, Trousseau University
Hospital, AP-HP, Paris, France, 8 Department of Genetics, Robert Debré
University Hospital, AP-HP, Paris, France, 9 Department of Genetics, Pitié-
Salpêtrière University Hospital, AP-HP, Paris, France, 10 Department of Medical
Genetics, Nantes University Hospital, Nantes, France, 11 Department of Medical
Genetics, Angers University Hospital, Angers, France, 12 Department of Medical
Genetics, Bordeaux University Hospital, Bordeaux, France, 13 Department of
Medical Genetics, Necker University Hospital, AP-HP, Paris, France.
Sotos syndrome is an overgrowth syndrome characterized by distinctive
facial features, learning difficulties and macrocephaly with frequent
pre- and postnatal overgrowth with advanced bone age. Here we report
on our experience in the molecular diagnosis of Sotos syndrome
on 116 patients. Using direct sequencing and a QMPSF-based assay
allowing accurate detection of both total and partial NSD1 deletions,
we identified NSD1 abnormalities in 104 patients corresponding to 102
Sotos families (90 %). NSD1 point mutations were detected in 80 %
of the index cases, large deletions removing entirely the NSD1 gene
in 14 % and intragenic NSD1 rearrangements in 6 %. Among the 69
detected distinct point mutations, 48 were novel. The QMPSF assay
detected an exonic duplication and a mosaic partial deletion. QMPSF
mapping of the 15 large deletions revealed the heterogeneity of the
deletions which size varies from 1 to 4.5 Mb. Clinical features of NSD1positive
Sotos patients revealed that the phenotype in patients with
non-truncating mutations was less severe that in patients with truncating
mutations. This study confirms the heterogeneity of NSD1 alterations
in Sotos syndrome and therefore the need to complete sequencing
analysis by the screening for partial deletions and duplications in
order to ensure an accurate molecular diagnosis of this syndrome.
22
P0868. Molecular analysis of SPG and SPG A mutations in
families with hereditary spastic paraplegia
C. Thieleke-Matos1 , L. Miller-Fleming1 , P. Magalhães1 , P. Coutinho2 , J. Sequeiros1,3
, I. Silveira1 , J. Loureiro1,2 ;
1 2 IBMC, Porto, Portugal, Hospital S.Sebastião, Santa Maria da Feira, Portugal,
3ICBAS, Porto, Portugal.
Autosomal dominant hereditary spastic paraplegias (AD-HSPs) are a
genetically and clinically heterogeneous group of neurodegenerative
disorders, characterized mainly by progressive weakness and spasticity
of the lower limbs. Overall, SPG4 and SPG3A mutations account
for about half of all AD-HSP cases. SPG4 encodes spastin, and is
associated with a pure phenotype, with no predictable age-at-onset,
whereas SPG3A encodes atlastin, being also associated with a pure
phenotype, but particularly distinguishable by its early onset and a
slower progression of symptoms. Recent data suggested that spastin,
an ATPase from the AAA protein family, and atlastin, an oligomeric
GTPase, are binding partners and function in the same biochemical
pathway. To genetically characterize Portuguese families with HSP,
80 unrelated patients, 61 with AD-HSP and 19 isolated cases were
analysed. Mutational analysis was performed by DHPLC followed by
sequencing. Twelve mutations in SPG4, nine of which novel ones, as
well as one novel mutation in SPG3A have been identified, so far. The
SPG3A mutation was a novel frameshift causing early onset (< 10
years) and a pure phenotype; all the patients presenting SPG4 mutations
had a pure phenotype; p.G370R was the only recurrent mutation
in Portuguese HSP patients. In conclusion, we genetically identified
21% of all AD-HSP in our group of Portuguese families. SPG4 mutations
had a frequency of 20%; together with the five other SPG4
families previously described, SPG4 accounts for approximately 28%
of known AD-HSP Portuguese cases.
P0869. SMN1 mutation analysis in 803 Spanish spinal muscular
atrophy patients.
L. Alias1 , M. J. Barceló1 , C. Hernández Chico2 , J. M. Millán3 , S. Alcover1 ,
I. Cuscó1 , E. Also1 , Y. Martín2 , L. Sáez2 , E. Aller3 , E. Galán4 , S. López5 , M.
Fernández Burriel6 , M. Baiget1 , E. F. Tizzano1 ;
1 2 Hospital St. Pau, Barcelona, Spain, Hospital Ramón y Cajal, Madrid, Spain,
3 4 Hospital La Fe, Valencia, Spain, Hospital Materno Infantil, Badajoz, Spain,
5 6 Hospital Virgen de la Candelaria, Tenerife, Spain, Hospital de Mérida, Badajoz,
Spain.
Spinal muscular atrophy (SMA) is an autosomal recessive disease
characterised by degeneration of the anterior horn cells of the spinal
cord and caused by mutations in the SMN1 gene. We present the results
of a systematic SMN1 gene analysis in 803 unrelated Spanish
SMA patients.
In 729 patients (91%) the molecular defect was absence of the
SMN1 gene and in 41 cases (5%) it was the presence of SMN hybrid
genes. We detected 24 subtle mutations (3%), 14 of which were
c.399delAGAG (representing 1.7% of the total Spanish SMA cases).
Two of these cases were homozygous for the mutation. By quantitative
PCR techniques, the remaining patients were heterozygous for
the SMN1 gene deletion. In the other allele we identified the following
mutations: p.Y272C (one case), p.T274I (one case) and 813ins/dup11
(two cases) described in other populations and p.I116F, p.Q136E,
c.738-740insC and c.867+2T>G described to date only in Spanish
SMA patients. Furthermore, two novel mutations, c.311-312insA (exon
3) and p.W190X (exon 4) were identified. In the remaining 9 patients
no changes were detected in the sequence of the codifying regions
and exon intron boundaries. mRNA was available in two of these patients
and no SMN1 transcripts were detected. Novel variants were
found in the promoter region of these patients. However, it is not possible
to ascribe these variants to the SMN1 or SMN2 promoter.
Employing this systematic approach, the SMN1 genotypes of the vast
majority of the Spanish SMA patients were characterised, including the
existence of non-functional SMN genes. FIS 05-2416.
P0870. Brain-derived neurotrophic factor (BDNF): mRNA
expression in rat brain focal ischemia under the treatment with
neuropeptides Semax and PGP
V. G. Dmitrieva 1 , L. V. Dergunova 1 , I. V. Vlasova 1 , O. V. Povarova 2 , V. I.
Skvortsova 2 , S. A. Limborska 1 ;
1 Institute of Molecular Genetics RAS, Moscow, Russian Federation, 2 Institute of