2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
P06.195
Blind assessment of High Resolution DNA melting Analysis as a
tool for mutation screening
S. Dwyer, N. M. Williams, N. Norton, M. J. Owen, M. C. O’Donovan;
Cardiff University, Cardiff, United Kingdom.
High resolution DNA melting analysis can be used to identify DNA
variants in PCR amplimeres . Using a LightScanner TM (Idaho Technologies)
we have developed a single analysis protocol that allows the
efficient screening of all PCR amplimeres by high resolution DNA melting
analysis to detect unknown DNA variants . In a blind assessment
of our protocol we screened 14 different amplimeres in 14 individuals
and identified 23/23 sequence variants, distinguishing 100% of those
where an individual was heterozygous for at least one site but only
57% of those individuals who were homozygous for a variant . In a
direct comparison, analysis using the Wave ® DHPLC system distinguished
96% of the heterozygous sequence changes and only 14%
of the amplimeres that differed by a homozygous sequence change .
Our data suggests that high resolution DNA melting analysis using the
LightScanner TM can be a highly sensitive mutation detection, which
because of its low cost and high speed could potentially allow more
comprehensive re-sequencing analysis of candidate genes .
P06.196
inter-laboratory diagnostic validation of conformation sensitive
capillary Electrophoresis.
C. J. Mattocks 1 , G. Watkins 1 , T. Janssens 2 , G. Matthijs 2 , E. Bosgoed 3 , K. van
der Donk 3 , H. Scheffer 3 , B. Pot 4 , J. Theelen 5 , T. Aspholm 6 , N. C. P. Cross 1 ;
1 National Genetics Reference Laboratory (Wessex), Salisbury, United Kingdom,
2 Center for Human Genetics (EuroGentest), Leuven, Belgium, 3 Dept. Human
Genetics, Radboud University, Nijmegen, The Netherlands, 4 Applied Maths,
Gent, Belgium, 5 Applied Biosystems, Europe, The Netherlands, 6 Applied Biosystems,
Europe, Norway.
Much of the work in diagnostic laboratories involves scanning for unknown
mutations in particular regions of interest . Whilst this work can
be done by sequencing, it is often faster and more economic to use an
indirect pre-screen to identify fragments containing variations . Such
methods need stringent validation to ensure diagnostic accuracy .
Conformation Sensitive Capillary Electrophoresis (CSCE) is a rapid
and sensitive method for indirect mutation scanning based on the principle
that homoduplex and heteroduplex DNA have different mobilities
when subjected to electrophoresis . We have performed a two phase
diagnostic validation of this method across three laboratories . Phase
I was used to define the performance characteristics of CSCE over a
range of critical parameters including the nature of the mutation, fragment
length and base composition, as well as a range of electrophoresis
variables . Phase II comprised a blinded and randomised investigation
of >400 different BRCA1 and BRCA2 variations to determine the
diagnostic accuracy of both CSCE itself and a Bionumerics software
plug-in used for the automated interpretation of heteroduplex patterns .
We will report the findings of these studies and discuss their general
applicability to diagnostic mutation scanning using CSCE .
P06.197
mYO9B polymorphisms in multiple sclerosis
A. Kemppinen 1 , M. Suvela 1 , P. Tienari 2 , M. Sumelahti 3 , I. Elovaara 4 , K.
Koivisto 5 , T. Pirttilä 6 , M. Reunanen 7 , P. Baumann 8 , J. Hillert 9 , F. Lundmark 9 , A.
Oturai 10 , L. Ryder 11 , H. Harbo 12,13 , E. Celius 13 , A. Palotie 14,15 , M. Daly 14 , L. Peltonen
1,15 , J. Saarela 1 ;
1 National Public Health Institute, University of Helsinki and FIMM, Institute
for Molecular Medicine Finland, Helsinki, Finland, 2 Department of Neurology,
Helsinki University Central Hospital and Neuroscience Programme, University
of Helsinki, Helsinki, Finland, 3 School of Public Health, University of Tampere,
Tampere, Finland, 4 Department of Neurology, Tampere University Hospital,
Tampere, Finland, 5 Central Hospital of Seinäjoki, Seinäjoki, Finland, 6 Department
of Neurology and Neuroscience, Kuopio University Hospital, Kuopio,
Finland, 7 Department Neurology, Oulu University Hospital, Oulu, Finland,
8 Department of Neurology, Lapland Central Hospital, Rovaniemi, Finland, 9 Division
of Neurology, Department of Clinical Neuroscience, Karolinska Institutet
at Karolinska University Hospital–Huddinge, Stockholm, Sweden, 10 Danish
Multiple Sclerosis Research Centre, Department of Neurology, Copenhagen
University Hospital, Copenhagen, Denmark, 11 Department of Clinical Immunology,
Copenhagen University Hospital, Copenhagen, Denmark, 12 Institute of Immunology,
University of Oslo, Oslo, Norway, 13 Department of Neurology, Ullevål
University Hospital, Oslo, Norway, 14 The Broad Institute of Harvard and MIT,
Cambridge Center, Cambridge, MA, United States, 15 Wellcome Trust Sanger
Institute, Cambridge, United Kingdom.
Multiple sclerosis (MS) is a complex disease manifesting as a chronic
inflammation in the central nervous system. It is also commonly classified
as an autoimmune disease (AID) and typical of many AIDs shows
strong linkage and association to the HLA locus . In addition to HLA,
AIDs are likely to share also other genetic risk factors . Recently, SNPs
in the 3’ region of the MYO9B gene have been reported to associate
with celiac disease, inflammatory bowel disease, rheumatoid arthritis
and systemic lupus erythematosus . The myosin IXB protein is an unconventional
myosin with a Rho-GTPase activity . It is involved in actin
cytoskeleton remodeling and is thereby a potential regulator of tight
junctions and epithelial and endothelial permeability . High expression
in leukocytes also suggests an immunological function . We tested the
association of MYO9B variants with MS in four Northern European
populations . Family-based association analyses (TDT, Gamete competition)
using 18 SNPs covering MYO9B in 730 Finnish MS families
showed no evidence for association . In order to increase the power to
detect variants with smaller effect size we further genotyped 11 SNPs,
2 of which have previously shown association to other autoimmune
diseases, in a set of 2511 MS patients and 2801 population controls
from Finland, Denmark, Norway and Sweden . However, no association
was observed in the case-control analysis, nor when stratifying for
the previously identified genetic MS risk factors at HLA and PRKCA
loci . Our results thereby do not support a major role for MYO9B variants
in multiple sclerosis .
P06.198
Functional analysis of missense mutations in the myocyte
Enhancer Factor 2A (mEF2A) gene do not support their causal
role in the pathogenesis of myocardial infarction
I. Guella 1 , V. Rimoldi 1 , P. A. Merlini 2 , R. Asselta 1 , E. M. Paraboschi 1 , M. Francolini
3 , F. Peyvandi 4 , D. Ardissino 5 , P. M. Mannucci 4 , S. Duga 1 ;
1 Dept. of Biology and Genetics for Medical Sciences, University of Milan, Milan,
Italy, 2 Niguarda Ca’ Granda Hospital, Milan, Italy, 3 Dept. of Medical Pharmacology,
University of Milan, Institute of Neuroscience - CNR - Cell. Mol. Pharmacology
Section, Milan, Italy, 4 A. Bianchi Bonomi, Hemophilia and Thrombosis
Center, University of Milan and Dept. of Medicine and Medical Specialties,
IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan,
Italy, 5 Maggiore Hospital, University of Parma, Parma, Italy.
Coronary artery disease (CAD) and its most severe complication
myocardial infarction (MI), are the leading causes of death in Western
countries . Mutations in the MEF2A gene, a member of the myocyte enhancer
factor-2 (MEF2) family of MADS-box transcription factors, have
been reported in patients with CAD/MI . In particular, a 21-bp deletion
and 3 missense mutations were demonstrated to affect MEF2A activity
either by reducing its transcriptional activity or by impairing its nuclear
translocation . However, the association of MEF2A with CAD/MI was
not confirmed by other studies.
In this work, we analyzed the role of MEF2A in the pathogenesis of
MI in a large cohort (1785 males, 223 females) of Italian patients with
premature MI (