2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.112
Evaluation of a PcR-based method that allows the analysis
of fragile X mutations in the complete range of expansions: a
multicentric italian experience
M. Grasso 1 , I. Giotti 2 , F. Torricelli 2 , M. Melis 3 , A. Loi 3 , A. Ravani 4 , A. Ferlini 4 , E.
Bettella 5 , R. Polli 5 , A. Murgia 5 ;
1 SC Laboratorio Genetica Ospedale Galliera, Genova, Italy, 2 SOD Diagnostica
Genetica AO Universitaria, Firenze, Italy, 3 Laboratorio Genetica Umana, ASL
8, Università di Cagliari, Cagliari, Italy, 4 Laboratorio Genetica Molecolare AO
Universitaria, Ferrara, Italy, 5 University of Padua Department of Pediatrics,
Padua, Italy.
We present the results of a collaborative study aimed at evaluating
the performances of the Fragile X PCR Assay (Abbot Molecular, Inc),
designed to provide robust amplification and precise sizing of FMR1
dynamic mutations within the entire range of expansions . The goal of
the study was to test the following features: 1) accuracy in measuring
the number of CGG repeats; 2) capacity to resolve normal FMR1 alleles
differing by one triplet unit, 3) capacity to assure efficient co-amplification
of different-size fragments; 4) amplification of full mutations.
The population studied comprised a total of 378 individuals, 241 females
and 137 males, all previously ascertained by Southern blot and
other types of PCR-based analysis which found 226 individuals with wt
FMR1 alleles, 80 with premutations, 48 with full mutations and 24 with
complex mosaicism . A few representative samples were analyzed by
all the laboratories involved . The study pointed out the importance of
evaluating the whole set of data generated through the analysis for a
correct interpretation of the final results. The work confirmed the sensitivity
and reliability of the method that allowed to correctly size FMR1
alleles in the different categories of expansions and obtained a robust
amplification of full mutations with up to 600 repeats. The Fragile X
PCR Assay is a molecular tool that can fill the existing gap between
currently used PCR-based methods that do not amplify large CGG
expansions and Southern blot analysis which does not allow a precise
sizing of FMR1 alleles .
P01.113
tissue mosaicism of unmethylated expanded FmR1 allele
derived from normal number of cGG repeats
C. Catalli1 , I. Bagni2 , A. M. Nardone2 , G. Fusco1 , M. Frontali2 , A. Morgante1 , A.
Botta1 , G. Novelli1,2 ;
1 2 Department of biopathology, Tor Vergata University, Roma, Italy, Medical
genetics, Policlinico Tor Vergata, Roma, Italy.
Fragile-X syndrome (FRAXA, OMIM #300624) is caused by expansion
of a [CGG] trinucleotide in the FMR1 gene over 200-230 repeats . This
leads to methylation of CpG islands of its promoter region, which in
turn causes silencing of FMR1 gene . Length and methylation mosaicisms
are described, arisen from premutated or mutated maternal alleles,
with different and often unpredictable clinical outcome . Here we
present the molecular characterization of a clinically normal 33 years
old man referred to us for genetic counseling after the identification
of a carrier status (61 [CGG]) of her daughter detected during amniocentesis
and confirmed at birth. Familial study revealed a maternal
transmission of the premutated allele . Surprisingly, a mosaic pattern
ranging from normal to 330 unmethylated FMR1 alleles was detected
in the father . On clinical re-evaluation, no sign associated with FRAXA
were detected, except relative macroorchidia and slightly extroverted
ears. Intelligence were above-average. Samples of fibroblasts, sperm
and saliva were additionally analyzed and all resulted normal . QF-PCR
and chromosome analysis excluded additional chromosome X mosaicism
. Blood sample of his mother showed the same normal allele, as
resulted from linkage analysis . To our knowledge, even if tissue-confined,
this is the first report of expanded FMR1 allele originating from
normal allele .
P01.114
Variation in novel exons (RAcEfrags) and human genetic
disorders; the case of Rett syndrome
P. Makrythanasis 1 , P. Kapranov 2 , L. Bartoloni 1 , A. Raymond 3 , S. Deutsch 1 , R.
Guigo 4 , F. Denoeud 4 , C. Rossier 1 , F. Ariani 5 , V. Capra 6 , A. Renieri 5 , T. Gingeras 2 ,
S. E. Antonarakis 1 ;
1 Medical Genetics and Dev, Medical School, University of Geneva, Geneva,
Switzerland, 2 Affymetrix, Santa Clara, CA, United States, 3 University of Lausanne,
Lausanne, Switzerland, 4 IMIM, Barcelona, Spain, 5 University of Siena,
Siena, Italy, 6 Neurochirurgia, Istituto G.Gaslini, Genova, Italy.
The study of transcription using genomic tiling arrays has lead to
the identification of numerous additional exons. One example is the
MECP2 gene on the X-chromosome; using 5’RACE and RT-PCR in
human tissues and cell lines, we have found more than 15 novel exons
(RACEfrags) connecting to at least one exon of MECP2 gene and
map up to 1 Mb telomeric to it . We subsequently asked if variation
in the novel exons is causatively associated with Rett syndrome . We
sequenced all MECP2-connected exons and flanking sequences in
3 groups: 48 Rett patients without mutations in MECP2 and CDKL5
genes (group_1); 30 Rett patients with mutations in the MECP2 gene
(group_2); 100 control individuals from the same geoethnic group
(group_3) . Approximately 14kb was sequenced per sample, (2 .6Mb
of DNA resequencing). 75 individuals had new, not yet identified rare
variants, but no statistically significant difference was found among the
3 groups . These results suggest that variants in the newly discovered
exons studied do not contribute to Rett syndrome, furthermore if some
of these variants are related to a phenotype, this must be different from
Rett . Interestingly however, the variants in the novel exons are twice
as frequent as those found in flanking sequences (50 vs 24 for approximately
1 .3 Mb sequenced for each class of sequences) . The significance
of this result remains to be elucidated; one hypothesis is that
novel exons accumulate variants faster than the rest of the genome
(positive selection?) that could underscore the functional importance
of these sequences .
P01.115
Rett syndrome cases with more than one causative mutation in
the mEcP2 gene
D. O. Robinson1,2 , D. J. Bunyan1 ;
1Wessex Regional Genetics Laboratory, Salisbury, Wilts, United Kingdom,
2Human Genetics Division, Southampton University School of Medicine, Southampton,
United Kingdom.
Rett syndrome is an X-linked dominant disease affecting females . It
is a progressive neurological disorder characterized by normal birth
and psychomotor development for the first six to 18 months of life followed
by severe regression in motor and language ability . In 80% of
cases mutations in the Methyl-CpG-binding protein 2 (MECP2) gene
are identified, usually de novo point mutations, however up to 16%
are caused by deletions of one or more exons of the gene . Almost all
are single cases in a family resulting from de novo mutations or the
inheritance of a mutation from an unaffected parent with somatic or
germ line mosaicism .
Point mutation testing and gene dosage analysis of a cohort of 455
cases referred to our laboratory for MECP2 gene analysis identified
mutations in 118 cases . However there were four females who each
had two different de novo causative mutations, presumed to be on the
same chromosome because compound heterozygosity for two causative
mutations is likely to be lethal . Two of these cases had a point
mutation and a small intraexonic deletion, a third had a whole exon
deletion and a separate small intraexonic deletion and a fourth had a
small intraexonic deletion and a large duplication. These findings highlight
the necessity to perform both point mutation and exon dosage
analysis in such cases, particularly because of the possibility of undetected
parental mosaicism and the implications for prenatal diagnosis
in future pregnancies . These cases also suggest that the MECP2 gene
may be particularly prone to mutation .
P01.116
FRAXA molecular Genetic Diagnosis of the oocyte´donor
population during 2007 for Assisted Reproduction treatments.
A. L. Garda-Salas 1 , I. Pérez 1 , K. Aurrekoetxea 2 , C. Méndez 1 , M. C. Martínez 1 ,
P. Albero 1 , L. Fernández 1 , M. Nicolás 1 , J. Landeras 1 ;
1 IVI-Murcia, Murcia, Spain, 2 Abbot Científica, Madrid, Spain.
The Instituto Valenciano de Infertilidad (IVI) has implemented for the
oocyte donation program in our clinics, the routine study for molecular
analysis of X-FRAGILE Syndrome (FRAXA) . FRAXA is one of the
greatest genetic prevalence illnesses in general population . In this
communication, we present our analysis, using the protocol recently
developed by Abbott Molecular, called Fragile X PCR Test, . We studied
2278 women from different cities in Spain . All these women as
susceptible oocyte’donors for processing of assisted reproductive
treatments (TRA) .