2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.090
Fragile X mosaic male detected by PCR/MS-MLPA
T. Todorov 1 , A. Todorova 1 , A. Kirov 2 , R. Carvalho 3 , I. Boneva 4 , V. Mitev 1 ;
1 Department of Chemistry and Biochemistry, Medical University, Sofia, Bulgaria,
2 Genetic Medico-Diagnostic Laboratory Genica, Sofia, Bulgaria, 3 MRC-Holland
bv, Amsterdam, The Netherlands, 4 Paediatric Hospital, Medical University, Sofia,
Bulgaria.
We report on a fragile X syndrome (FXS) mosaic male full mutation/
normal allele, detected by a combination of PCR and methylation-specific
multiplex ligation-dependent probe amplification (MS-MLPA). This
analysis provides a powerful, fast, cheap and easy to perform diagnostic
approach for FXS. This is the first report on successful application
of MS-MLPA for FXS diagnostic purposes . The combination of PCR
and MS-MLPA gives the possibility in a few steps to detect normal
FMR1 alleles, to prognose the expanded ones, to assess the CpG
islands methylation, as well as to determine copy number changes like
large deletions/duplications, not only along the FMR1, but also along
the FMR2 gene .
Our PCR results showed one allele of 29±1 repeats in the mother and
one allele in the affected boy, but three repeats larger - 32±1 repeats .
The MS-MLPA results in the patient showed hypermethylated full mutation
pattern in comparison to the normal control . The MS-MLPA data
calculations were performed in Excel .
In our opinion, the mosaic pattern of normal size/full mutation alleles
was a result from inheritance of a maternal unstable premutated allele .
Most logical mechanism for normal size allele generation in our mosaic
case is a deletion of a portion of the full mutation, restricted to the CGG
repeat itself, as the primers for PCR were designed in the repeat flanking
regions . The reported patient demonstrates atypical mild clinical
manifestation of the disease, which might be due to the presence of a
normal size allele in a large percentage of the patient’s cells .
P01.091
No evidence for skewed X-inactivation in fragile X syndrome
premutation carriers
C. Berenguer 1,2 , L. Rodriguez-Revenga 3 , I. Madrigal 3 , C. Badenas 1 , M. Milà 1 ;
1 Biochemistry and Molecular Genetics Department, Hospital Clínic, Barcelona,
Spain, 2 Fundació Clínic, Barcelona, Spain, 3 Centre for Biomedical Research on
Rare Diseases (CIBERER), ISCIII, Barcelona, Spain.
X-Chromosome inactivation (XCI) is the mechanism by which gene
dosage equivalence is achieved between female (XX) and male (XY)
mammals . In the general female population, the X-inactivation process
is random, and therefore XCI ratios have a normal distribution (with
average of 50:50) with only a small percentage of females (5-8%)
showing a skewed X-inactivation ratio (>90:10) . Fragile X syndrome
(FXS) premutation carriers (55-200 CGG repeats) do not present FXS
symptoms but it has been shown that they have a higher risk of developing
premature ovarian failure (POF) and/or fragile X associated
tremor/ataxia syndrome (FXTAS) . About 20% of the FXS female premutation
carriers present POF and around 15% FXTAS . In order to
evaluate if these pathologies are associated with skewed XCI patterns,
we have studied the X-inactivation pattern in 270 FXS females carriers
(41 POF, 2 FXTAS, 3 POF and FXTAS, and 224 no POF no FXTAS) .
Results showed that FXS permutation female carriers have a normal
distribution and that there is no correlation with the CGG repeat number
. On the basis of these observations we conclude that FXS female
premutation carriers with or without POF and/or FXTAS do not present
skewed X-inactivation and therefore, other molecular or environmental
factors may predispose to these conditions .
Acknowledgments: Marató TV3 (TV06-0810) and SAF- 2004-03083
P01.092
High conservation of the 3’UtR of FMR at potential microRNA
target sites in patients with fragile X syndrome premutation
L. Rodriguez-Revenga1 , M. Santos2,3 , I. Madrigal1 , C. Berenguer2,3 , X. Estivill4,5,6
, M. Mila3 ;
1 2 3 CIBERER, Barcelona, Spain, Fundació Clínic, Barcelona, Spain, Hospital
Clínic, Barcelona, Spain, 4Center for Genomic Regulation, Barcelona, Spain,
5 6 National Genotyping Center, Barcelona, Spain, Pompeu Fabra University,
Barcelona, Spain.
Fragile X syndrome premutation condition consists of an intermediate
CGG repeat expansion (55-200 repeats) at the 5’UTR of the FMR1
gene . In the premutation range, FMR1 mRNA levels are increased
whereas the amounts of FMRP are slightly reduced compared with
the control population, suggesting that posttranscriptional regulation of
FMR1 could be involved in these disorders . MicroRNAs act as regulators
of gene expression binding to their target sites located at the
3’UTR in a high number of protein-coding genes inducing cleavage
or repression of translation . We have screened the 3’UTR of FMR1
in 40 Mediterranean premutation carriers and 14 control subjects with
no expansion in the FMR1 gene . Overall, the FMR1 3’UTR region appears
as a high conserved region, not only in humans, but also in other
species . Our study excludes changes in the FMR1 3’UTR in the premutated
patients , ruling out a possible role of microRNA target sites
in FMR1 regulation in permutated phenotypes . Acknowledgements
(SAF2004-03083, Marató TV06-0810)
P01.093
molecular and epigenetic characterization of FMR
unmethylated full mutation cell lines
E. Tabolacci 1 , M. Accadia 1 , L. Borrelli 1 , M. Moscarda 1 , F. Zalfa 2 , C. Bagni 2 , U.
Moscato 3 , P. Chiurazzi 1 , G. Neri 1 ;
1 Institute of Medical Genetics, Rome, Italy, 2 Department of Biology, “Tor Vergata”
University, Rome, Italy, 3 Institute of Hygiene, Catholic University, Rome,
Italy.
Fragile X syndrome (FXS) is mostly caused by expansion and subsequent
methylation of the CGG repeat at the 5’ UTR of the FMR1 gene
(methylated full mutation) . Individuals of normal intelligence, carriers
of a FMR1 unmethylated full mutation, represent rare exceptions . We
previously performed a molecular and epigenetic analysis of a lymphoblastoid
cell line (code 5106), derived from one of these individuals
. Recently, two apparently normal individuals with an unmethylated
full mutation, belonging to distinct FXS families, were identified. From
each subject (named DPM and MA) three independent lymphoblastoid
cell lines and a fibroblast colture (from MA) were established. In accordance
with our previous findings, these cell lines showed normal
transcription and reduced translation of the FMR1 gene, compared to
normal controls . Epigenetic analysis of the FMR1 locus demonstrated
lack of DNA methylation and the methylation pattern of lysines 4 and
27 on histone H3 was also similar to that of a normal control, in accordance
with the normal transcription and consistent with an euchromatic
configuration. On the other hand, the H3 and H4 acetylation and
the methylation of lysine 9 on histone H3 was similar to that of a typical
FXS cell line . Comparative analysis of these rare unmethylated full
mutation cell lines demostrates remarkable structural, functional and
epigenetic consistency, suggesting a common mechanism of origin,
genetically determined . The discovery of such mechanism may be important
in view of therapeutic attempts to convert a methylated to unmethylated
full mutation, restoring the expression of the FMR1 gene .
P01.094
Analysis of fragile-X premutation and grey zone in paediatric
patients
L. Martorell 1 , P. Poo 2 , M. Ferrando 2 , M. Naudo 1 , J. Genovés 1 , A. Sans 2 ;
1 Molecular Genetics Section. Hospital Sant Joan de Déu, Barcelona, Spain,
2 Department of Neurology. Hospital Sant Joan de Déu, Barcelona, Spain.
INTRODUCTION: The number of CGG repeats defines the state of
premutation or full mutation in fragile-X syndrome (FXS) . In childhood,
full mutation has a characteristic behaviour and physical phenotype .
The adult carriers of the premutation can develop a progressive neurodegenerative
syndrome (FXTAS) . Although the clinical characteristics
of the premutation and grey-zone in the childhood are less well-known,
clinical experience and case reports suggest that child with premutation
and grey-zone alleles may have similar clinical features than those
with the full mutation .
PATIENTS AND METHODS: Determination of the number of CGG repeats
in samples received in our unit since 2005 for the FXS screening
.
Detailed Clinical and cognitive evaluation in a series of 13 children (9
boys and 4 girls), born between 1989 and 2000, with clinical suspect
of FXS .
RESULTS: From the 1092 samples analyzed, 7% (77 cases) we detected
alleles with 35 to 160 CGG repeats . We haven’t detected any
alleles in the permutated or grey-zone in the 91 control chromosomes
analyzed . The 13 children evaluated clinically have a cognitive-behavioural
phenotype suggestive of full mutation and we detected alleles