2008 Barcelona - European Society of Human Genetics

Clinical genetics
other CNPs, the 1q42 .12 region contains the ENAH gene that represents
a good candidate as RTT modifier since it . is involved in the
pathways that control cortical neuronal positioning . Real-time qPCR
with specific probes for selected candidate genes in 100 classic and
20 PSV patients is ongoing. The identification of modifier genes will
allow to better characterize the pathogenic mechanisms of RTT, giving
additional handles for therapy design .
P01.108
Rett syndrome in two years old girl with Xp deletion - case
report
A. Todorova 1 , T. Todorov 1 , R. Tincheva 2 , D. Avdjieva 2 , I. Boneva 2 , V. Mitev 1 ;
1 Department of Chemistry and Biochemistry, Medical University Sofia, Sofia,
Bulgaria, 2 Paediatric Hospital, Medical University Sofia, Sofia, Bulgaria.
Rett syndrome (RTT; OMIM#312750) is an early childhood neurodevelopmental
disorder . A girl at age of 2 years was referred to our laboratory
for DNA analysis of Rett syndrome . She demonstrated some
of the symptoms, characteristic for Rett syndrome - arrested mental
development, loss of communication skills, speech delay and purposeful
hand movements, appearance of autistic features . Under some circumstances
the child demonstrates aggressive behavior .
The performed cytogenetic analysis showed pathological karyotype -
46,XX,del(X)(p1 .22) .
About 85% of the cases with Rett syndrome were caused by mutations
in the gene encoding methyl-CpG-binding protein 1 (MECP2) . In about
10% of the cases the disease causing mutation affects the gene for
cyclin dependent kinase like 5 (CDKL5) . However, the causative mutation
is still unknown in the remaining 5% of the cases .
The complete sequencing of MECP2 and CDKL5 genes revealed no
mutations in the affected girl . In addition we sequenced the Aristaless
related homeobox (ARX) gene, mutations in which have been shown
to cause mental retardation either isolated or associated with a broad
spectrum of neurological problems . This gene has been mainly affected
in boys . As this gene is localized on the short arm of the X-chromosome
(Xp22 .13), one of its copies was missing in our Xp deleted girl .
For that reason this gene seemed to be a good candidate for screening
in our mentally retarded girl . The DNA analysis of ARX showed no
pathological changes .
We will appreciate any further comments and suggestions on the reported
case .
P01.109
ARX mutations in south African patients with X-Linked mental
retardation (XLmR): Research to diagnostics
G. L. Carvill, R. G. Goliath;
Division of Human Genetics, Institute of Infectious Disease and Molecular
Medicine, Cape Town, South Africa.
Objectives: Mental retardation (MR) is associated with decreased
cognitive function and impaired development of adaptive skills . The
genetic heterogeneity of the disorder is exemplified by the number of
genes implicated in the pathogenesis of MR . Of these genes, a substantial
proportion are located on the X chromosome, giving rise to the
term X-linked mental retardation (XLMR) . Mutations in the ARX gene
are the second largest contributor to XLMR, preceded only by CGG
expansion mutation in FMR1, responsible for Fragile X syndrome .
methods: DNA sequence alterations in the ARX gene were investigated
in 119 XLMR patients, 32 patients that form part of a male
sib-ship and 183 isolated cases (all individuals are FMR1 expansion
mutation negative) . These analyses were conducted using denaturing
high-performance liquid chromatography (dHPLC), or PCR amplification
and gel electrophoresis of the common c .428_451dup (Dup24)
mutation alone .
Results: To date the proportion of ARX disease-causing mutations in
the XLMR cohort is 2 .4% and 3,4% in the sib-ships . While in the isolated
group the Dup24 mutation has not been detected .
conclusion: These findings suggest that c.428_451dup testing is feasible
and justified in determining the cause of XLMR in males negative
for Fragile X syndrome, in the Western Cape region of South Africa . Incorporating
this test into the diagnostic protocol stands to improve the
diagnostic yield of patients, which in turn will afford improved genetic
management of families . Furthermore, better genetic management will
have a valuable impact on the burden of disease in a developing country
such as South Africa .
P01.110
molecular diagnostic of XLmR in mentally retarded males from
Latvia
Z. Daneberga1,2 , Z. Krumina1 , B. Lace1,2 , D. Bauze1 , N. Pronina1 , R. Lugovska1 ;
1 2 Medical Genetic Clinic, University Children`s Hospital, Riga, Latvia, Rigas
Stradins University, Riga, Latvia.
Mental retardation (MR) is one of the main reasons for referral in paediatric,
child neurological and clinical genetic practice . The prevalence of mental
retardation is thought to be on the order of 2-3% .
X-linked gene defects have long been considered to be important causes of
mental retardation, on the basis of the observation that mental retardation
is significantly more common in males than in females. Clinical observations
and linkage studies in families revealed that X-linked mental retardation
(XLMR) is a highly heterogeneous condition . The most common form
of XLMR is the Fragile X mental-retardation syndrome (FXS) . Mutations at
FRAXA locus on distal Xq may cause mental impairment . Most common
mutation at FRAXA locus is expansion of CGG triplet repeats located in the
5’-untranslated region of the fragile X mental retardation-1 (FMR1) gene .
The group of 341 unrelated males with MR referred from clinical geneticists
was screened for FXS . CGG repeats number was detected by Applied Biosystems
protocol on ABI Prism 310 . The prevalence of 29, 30 and 31 CGG
repeats for normal alleles were found . Six affected patients were detected
(1.76%). The final diagnosis of FXS was confirmed by Southern blotting.
DNA sequencing for the estimation of AGG inserts structure for gray zone
(34-50 repeats) alleles was used .
To analyse undiagnosed XLMR patients we recently introduced the multiplex
ligation-dependent probe amplification (MLPA) method to identify deletions
and duplications of MRX genes . We use MLPA P106 MRX commercial
kit (MRC-Holland) to analyse one or more exons of 14 MRX genes .
P01.111
Novel PcR-based protocol for a rapid molecular testing of
Fragile X syndrome
B. López-Posadas 1 , E. Velasco 2 , J. A. Garrote 3,4 , M. Alonso 1 , J. J. Telleria 1 , A.
Blanco 1 , I. Fernández-Carvajal 5 ;
1 Laboratorio de Genética Humana, Unidad de Diagnóstico Genético y Perinatal,
Valladolid, Spain, 2 Laboratorio de Genética del Cáncer, Unidad de Diagnóstico
Genético y Perinatal, Instituto de Biología y Genética Molecular (IBGM),
Universidad de Valladolid-CSIC, Valladolid, Spain, 3 Unidad de Investigación,
Hospital Clínico Universitario de Valladolid, Valladolid, Spain, 4 Laboratorio de
Inmunología, Unidad de Diagnóstico Genético y Perinatal, Instituto de Biología
y Genética Molecular (IBGM), Universidad de Valladolid-CSIC, Valladolid,
Spain, 5 Laboratorio de Genética Humana, Unidad de Diagnóstico Genético y
Perinatal, Instituto de Biología y Genética Molecular (IBGM), Universidad de
Valladolid-CSIC, Valladolid, Spain.
Fragile-X syndrome (FXS) is the most common cause of inherited
mental retardation . It is caused by an anomalous expansion of CGG
repeats in the 5’UTR of the FMR1 gene, which are hypermethylated,
causing the absence of FMR1 expression .
Clinical diagnosis of typical FXS is usually difficult to establish before
puberty and molecular testing is therefore needed to confirm it.
Several PCR protocols have already been developed to amplify these
repeats, but neither of them is able to amplify full-mutated alleles .
With a view to improving the molecular analysis of FXS, we have established
a new PCR-based strategy .
We analyzed 68 retrospective samples of known repeat sizes to assess
protocol efficiency and 252 prospective samples.
Two PCRs were performed followed by electrophoresis in a DNA sequencer.
Finally, a methylation-specific PCR was carried out to test the
promoter methylation status .
Results from retrospective samples were reproduced with the new protocol,
supporting its capability to test the whole range of FMR1 alleles .
Additionally, its sizing accuracy made allele frequency distribution and
transmission studies possible .
Analysis of prospective samples revealed 13 full mutations, two premutation-full
mutation mosaicisms, 6 premutations and 10 grey-zone
patients .
Moreover, we have amplified and accurately sized a full-mutated allele
of 817 repeats, the longest allele amplified by PCR until now.
In conclusion, this PCR approach has improved and speeded up the
FXS diagnosis making it less labor-intensive than standard procedures
that include Southern blot analysis .