2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
P11.075
Experimental and critical assessment of six methodological
approaches to quantify heteroplasmy of mitochondrial
mutations
I. Kurelac, M. Lang, R. Zuntini, G. Gasparre, G. Romeo;
Medical Genetics, S. Orsola-Malpighi Hospital, University of Bologna, Bologna,
Italy.
Mitochondrial DNA (mtDNA) mutations have an important effect on the
development of many genetic diseases, and are believed to play a role
in aging and cancer. Most mammalian cells contain hundreds of mitochondria
and each mitochondrion is endowed with several copies of
mtDNA. However, an individual’s mitochondrial asset may not always
be uniform. After an mtDNA variation arises, it may be maintained, lost
or amplified to different levels. As a result, cells and tissues, but also
single mitochondria, may harbor both wild-type and mutant mtDNA, a
condition known as heteroplasmy. In order for an mtDNA mutation to
show a phenotypic effect, a certain threshold of heteroplasmy must be
reached. Therefore, it is extremely important not only to qualitatively
detect a mutation but also to provide an adequate and efficient quantitative
analysis of heteroplasmy. Here we compare six different methodologies
for their capacity to evaluate, capture and quantify different
heteroplasmy levels. We also considered the issue of the potential
bias introduced by preferential amplification during polymerase chain
reaction (PCR). The mutation we chose to investigate is an insertion
of a single base in ND1 gene described by Bonora et al (Cancer Res,
2007). Cloning and sequencing was set as the golden standard and
the comparison was made between the following methods: fluorescent
PCR, denaturation high performance liquid chromatography (DHPLC),
quantitative real-time PCR (qRT-PCR), high resolution melting analysis
(HRM) and 454 pyrosequencing. The final results will be presented.
P11.077
in-lane Normalization improves the Analysis of mLPA Data
Obtained from capillary Electrophoresis systems
E. Schreiber1 , L. Pique2 , C. Davidson1 , E. Nordman1 , B. Johnson1 , R. Fish1 , L.
Joe1 , A. Pradhan1 , A. Felton1 , I. Schrijver2 ;
1 2 Applied Biosystems, Foster City, CA, United States, Stanford University Medical
Center, Stanford, CA, United States.
Multiplex ligation-dependent probe amplification (MLPA®, MRC-Holland)
is an OLA-PCR- (oligonucleotide ligation assay PCR) based
method used predominately for detecting copy number changes, such
as whole exon deletions and duplications, in gDNA. Typically, MLPA
reactions are analyzed on capillary electrophoresis (CE) instruments
and specialized secondary analysis software is used to normalize
the data and to calculate probe ratios to determine the presence of
duplications or deletions. Here we describe the use of a new in-lane
normalization reagent that is added to MLPA sample reactions prior to
CE analysis. The data collection software on a newly developed CE
instrument calculates a normalization factor that can then be applied
to the raw MLPA data by the GeneMapper® v4.1 secondary analysis
software. To demonstrate the benefits of in-lane normalization to MLPA
data, gDNA samples for Pendred syndrome patients were analyzed
using an MLPA kit (P280 Pendred-SLC26A4, MRC-Holland) designed
to interrogate the causative gene. Pendred syndrome, the most common
syndromal form of deafness, is an autosomal recessive disorder
associated with developmental abnormalities of the cochlea, sensorineural
hearing loss, and diffuse thyroid enlargement. Pendred syndrome
results from mutations in the Solute Carrier Family 26, Member
4 (SLC26A4) gene. The MLPA multiplex kit consists of probes for
each of the 21 exons of SLC26A4 in addition to two mutation-specific
probes. We have found that in-lane normalization significantly improves
the reproducibility and robustness of MLPA data analysis when
using GeneMapper v4.1 software.
P11.078
Generation of creER-t2 transgenic mouse lines for temporal
and cell type specific conditional gene inactivation: a tool for
analysis of pathomechanisms in mouse models of monogenic
diseases
L. Venteo 1 , N. Chartoire 1 , F. Augé 1 , J. Gallego-llamas 1 , M. Koch 1 , G. Neau 1 , N.
Ott 1 , F. Gofflot 1,2 , X. Warot 1 , J. Auwerx 3 , J. L. Mandel 3 , M. C. Birling 1 , G. Pavlovic
1 ;
1 Institut Clinique de la Souris, Illkirch, France, 2 univ catholique louvain, Louvain
la neuve, Belgium, 3 Institut Clinique de la Souris, IGBMC, Illkirch, France.
The generation of mouse mutants by conventional knock-out shows
two major limitations: (i) gene disruption often results in lethal phenotypes
(ii) it does not allow site specific and time controlled inactivation
of the gene of interest. Conditional knock-outs overcome these limitations.
In the Cre-loxP system, the allele of interest is flanked by recognition
sites for the Cre recombinase (loxP sites). When “floxed” mice
are bred with transgenic mice expressing Cre in a tissue/cell-specific
manner, the gene is knocked-out only in this tissue or cell. Temporal
control is achieved using a ligand-activated recombinase, a fusion of
the recombinase with a mutated ligand binding domain of the estrogen
receptor (ER), which can only be activated by the synthetic ligand
tamoxifen (Cre ERT2).
At ICS, we have generated about 50 Cre lines expected to express
CreERT2 recombinase in different target tissues or cells. These include
different neuronal populations, adipose tissue, different cell
populations in the digestive tract, pancreas, muscle, bone, immune
system, reproductive tract, skin ... Characterization of the efficacy and
specificity of such lines is demanding, and we have devised a standardized
flow-scheme (F. Gofflot et al.). Various lines are at different
stages of characterization. We will provide the list of promoters and
targeted tissues, and an update on their validation. These lines will
be available to the research community and will be a powerful tool for
the study of disease genes function, creation of disease models and
to answer questions on the cell/organ autonomous or not character of
pathological phenotypes.
P11.079
New method and new tool for human mtDNA phylogeny
reconstruction
N. Eltsov, N. Volodko;
Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russian Federation.
Human mtDNA sequence variation is characterized by a high amount
of homoplasy which does not always allow unequivocal phylogeny reconstruction
by conventional methods. We propose a novel maximum
parsimony-based method for reconstruction of human mtDNA phylogeny.
Briefly, the method consists of three successive stages: identification
of potential homoplastic mutations; analysis of potential homoplastic
mutations and identification of true homoplastic mutations;
identification of back and parallel mutations. The method designed
was implemented in mtPhyl. This software package allows rapid and
comprehensive analysis oh human complete mtDNA sequences. Apart
from maximum parsimony phylogenetic tree reconstruction it performs
different types of searches; analyzes mutation features; exports list of
particular mtDNAs mutations into Excel table; defines mitochondrial
haplotype; calculates coalescence time of clusters; estimates the effect
of natural selection; makes reference list and downloads human
mtDNA complete sequences from GenBank. mtPhyl represents a
timely advance, since the advent of cheaper sequencing methods has
generated an excess of sequence data, and there is an urgent need to
perform their automatic analysis. Demo version of mtPhyl is available
from the authors upon request and at http://eltsov.org/mtphyl.aspxl.
P11.080
Describing complex sequence variants by extending HGVs
sequence variation nomenclature
P. E. M. Taschner, J. T. den Dunnen;
Center for Human and Clinical Genetics, Leiden, The Netherlands.
New technologies allow rapid discovery of new sequence variants involving
complex structural rearrangements. The description of these
variants challenges the existing sequence variation nomenclature
guidelines of the Human Genome Variation Society (HGVS, http://www.
hgvs.org/mutnomen), which are mainly focused on simple variants.
Here, we suggest extending the HGVS nomenclature guidelines with
new description formats facilitating unambiguous and more detailed
descriptions of most complex sequence variants. These include: 1)
nested changes supporting descriptions of changes within inversions
and duplications, 2) composite changes supporting concatenation of
inserted sequences, 3) new duplication types describing changes in
orientation. One advantage of this extension is that the differences
and similarities between complex variants can be derived easily from
the new descriptions. In addition, this extension is expected to provide
sufficient flexibility and consistency to limit the proliferation of alterna-