2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
scientific reporting in general, needs to be developed.
At the heart of this lies the concept of a user-centric system for researcher
identification - i.e., one or more ‘ID systems’ by which individuals
can be unambiguously identified along with various types of
information associated with them, and where the individual controls
his/her online identity and how/where it is used. At present, key Web
2.0 Internet technologies which can underpin such a system (e.g.,
OpenID - a decentralized, open authentication protocol), are being
widely adopted.
To advance this field, a community of key stakeholders (e.g., GEN-
2PHEN, P3G, HUGO, HVP) has been assembled and is continually
growing. This group is exploring innovative ways to exploit this new
Internet ecosystem to support research-related activities and services.
A dedicated website is provided (via www.gen2phen.org) where issues
are being discussed, and a workshop on the subject has been planned
for May 2009.
Acknowledgements: GEN2PHEN is funded by the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant
agreement 200754.
P11.105
Abundance and orientation bias of retroelements in mammalian
genes
N. V. Tomilin, N. V. Tomilin;
Russian Academy of Sciences, St.Petersburg, Russian Federation.
In different eukaryotic genomes some transposable elements (TEs)
located in introns of genes show significant bias with predominant
antisense orientation of TEs. It was suggested that this bias can reflect
negative selection against TEs in the orientation of coding strand.
In mammalian genomes retrolelements of L1 family are known to be
strongly underrepresented in GC-rich gene rich regions but their abundance
and orientation in introns of genes is poorly studied. Here we
analyzed abundance and orientation of major families of retroelements
in introns and promoters in eight large groups (>300 genes each) of
coexpressed human and mouse genes. We found that human Alu
and mouse B1 repeats are overrepresented in introns and upstream
regions of all studied groups of genes with significant bias for their
inverse orientation in groups of the widely expressed (housekeeping)
genes. L1 elements are underrepresented in all studied groups of
genes especially in the upstream regions and only ~30% of L1 is found
in direct orientation. LTRs are also underrepresented in all groups of
human and mouse genes with only 15-30% of their copies present
in direct orientation suggesting strong selection pressures operating
during evolution on retroelement content of promoters and introns of
human genes. Purifying selection of LTRs, L1 elements and some Alu
repeats in direct orientation is apparently a consequence of their negative
effects on normal transcription and/or splicing. Accumulation of
Alu and B1 in the housekeeping and some tissue-specific genes may
be caused by positive selection of complex genome rearrangements
facilitating transcription.
P11.106
MeCP gene point mutation analysis, gross deletions and Xchromosome
inactivation in 200 girls with Rett syndrome and
with variants of the disease in Russia.
O. V. Babenko 1,2 , V. V. Strelnikov 1,2 , T. V. Kekeeva 2 , G. G. Guzeev 3 , V. G.
Solonichenko 3 , N. A. Demina 1 , V. A. Galkina 1 , G. E. Rudenskaya 1 , D. V. Zaletayev
1,2 ;
1 Research Centre for Medical Genetics, Moscow, Russian Federation, 2 Institute
for Molecular Medicine, Moscow Medical Academy, Moscow, Russian Federation,
3 Centre for Medical Genetics, N.F.Filatov Pediatric Hospital, Moscow, Russian
Federation.
Rett syndrome (RTT) is a neurodevelopmental disorder inherited in an
X-linked dominant manner affecting almost exclusively females. MeCP2
gene (Xq28) is responsible for both classic and atypical cases of Rett
syndrome. Mutation analysis of MeCP2 gene was performed in 200 female
patients with classic and variant phenotypes of RTT and with nonspecific
mental retardation by using SSCP analysis and sequencing of
the coding region of the MeCP2 gene. Prior to mutations screening we
have excluded Angelman syndrome (methylation in 15q11-q13 region),
Smith-Magenis syndrome (17p11.2 deletion) and CGG expansion of
FMR1 in patients with nonspecific mental retardation.
More mutations in this study have been identified in 64% patients with
classical forms (half of these mutations were revealed in 8 hot spots
of MeCP2 gene) of Rett syndrome compared to atypical patients with
mental retardation (28%).
Gross rearrangements of the MECP2 gene, which are not detectable
by sequencing or SSCP, have been identified using dosage assays
including quantitative fluorescent PCR and real-time PCR. Large deletions
were identified in 15% cases of our patients in whom no MECP2
mutation had previously been detected by sequence analysis.
Variability of Rett phenotype has been partly attributed to an effect of
X-chromosome inactivation. In the same cohort of patients skewed Xinactivation
was detected in 10% RTT cases. Moreover, phenomenon
of nonrandom X-inactivation (NXI) assessed by CAG polymorphism
analysis of AR (HUMARA) gene was detected in mothers of RTT patients
(38%). In these cases NXI was found in the absence of mutation
and clinical features of diseases.
P11.108
some points for the optimization of the semiquantitative
multiplex PcR assay for detection of exon rearrangements in the
Parkin gene
Z. Fazlali 1,2 , F. Ghazavi 1 , S. S. Banihosseini 3 , S. Shojaee 4 , E. Elahi 1,5 ;
1 School of Biology, College of Science, Tehran, Islamic Republic of Iran, 2 National
Elite Foundation, Tehran, Islamic Republic of Iran, 3 Tehran University of
Medical Sciences, Tehran, Islamic Republic of Iran, 4 Department of Biotechnology,
College of Science, Tehran, Islamic Republic of Iran, 5 Center of Excellence
in Biomathematics, School of Mathematics, Statistics and Computer Science,
College of Science, University of Tehran, Tehran, Islamic Republic of Iran.
Mutations in the parkin gene are responsible for a notable fraction of
autosomal recessive early onset Parkinson’s disease incidence worldwide.
Exon rearrangements in the gene constitute an important class of
the mutations. In our study we performed a commonly used semiquantitative
multiplex PCR assay for detection of rearrangements of Parkin
exons. The assay essentially compares relative amounts of template
DNA in a PCR reaction, thus allowing identification of templates that
deviate from the expected number in a diploid genome. In order to
detect these rearrangements, several exons are PCR amplified simultaneously,
allowing comparison of relative amplification of exons in different
samples. The co-amplified exons serve as internal standards
for quantification. The amount of PCR product is directly related to the
number of template molecules as long as PCR is terminated within the
exponential phase of amplification. Each PCR for a given combination
of exons results in a typical pattern of peak heights for normal control
DNA, thus producing reference ratios between the peaks. Because
rations are compared, extraneous factors are expected not to interfere
with detection of rearrangements.
Our study resulted in identification of several exon deletions in the Parkin
gene. We noted that for optimal detection of rearrangements, careful
attention must be made to several factors: -all the fluorescently-labeled
primers in a combination should be labeled with the same dye;
-height of exon peaks reflecting extent of amplification should be very
similar for the different exons (less than 10% difference); -quality of the
DNA samples should be high.
P11.109
High coverage, low bias libraries for the sOLiD 3 system to
support 2x50 mate pair libraries
M. D. Rhodes1 , K. Varma1 , B. Li1 , Z. Liu1 , E. Gerdts1 , D. Greiner1 , J. Ziegle1 , C.
Clouser2 , G. C. Costa2 , K. Mckernan2 , T. Burcham1 , A. Shah1 ;
1 2 Applied Biosystems, Foster City, CA, United States, Applied Biosystems,
Beverly, MA, United States.
Paired end sequencing facilitates sequence assembly and broadens
the application of next generation sequencing technologies. Library
construction strategies that utilize type III restriction enzymes, such
as EcoP15I and MmeI, generate mate-paired libraries with limited sequence
tag length. As the read length of the SOLiD system improves,
a library construction method that generates longer DNA mate pairs is
necessary. We describe a paired end library construction method that
uses nick translation activity of DNA polymerases and T7 exonuclease
to generate DNA mate pairs. The length of the DNA mate pairs can be
adjusted by controlling the temperature and time of the nick translation
reaction. We have prepared multiple paired end libraries with tag
lengths of 75-100bp using many complex genomes. These libraries
have been extensively sequenced at 50 bp for each tag using SOLiD,
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