2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
sequence are avoided during primer design . The resulting primers are
then checked for genomic redundancy and the probability of success
in PCR . An exhaustive search is performed to produce the optimal tiling
of the amplicons covering the target region . Utilization of this primer
design tool can substantially reduce the effort required to design and
optimize robust resequencing primers .
P08.68
Prescreen for RB mutation identification using high-resolution
melting analysis
E. K. Zhang 1 , D. Rushlow 1 , N. L. Prigoda 1 , B. Piovesan 1 , K. Vandezande 1 , B.
L. Gallie 1,2 ;
1 Retinoblastoma Solutions, Toronto, ON, Canada, 2 The Hospital for Sick Children,
The Vision Science Research Program, University Health Network and
Departments of Ophthalmology, Molecular and Medical Genetics and Medical
Biophysics, University of Toronto, Toronto, ON, Canada.
Germ-line mutations in the RB1 gene cause hereditary predisposition
to retinoblastoma, a childhood eye cancer, but 90% of the tumors are
caused by novel mutations . Since the RB1 gene spans 180 kb and
contains 27 exons, and RB1 mutations have been observed in all 27
exons and promoter with few recurrent mutations, multiple modalities
are required in addition to sequencing to attain high sensitivity for mutation
detection . Amplicon melting with a saturating DNA intercalating
dye was introduced as an attractive technique to genotype small sequence
alterations. In order to investigate the efficiency of high-resolution
melting analysis (HRM) in RB1 mutation screening, we tested its
sensitivity to detect known variants .
PCR products were prepared for routine sequencing . Resolight dye
master mix was added post-PCR . Fluorescence melting curves were
recorded using the LightCycler 480 . All (10/10) heterozygous and 2/2
homozygous variants were readily discriminated from wild-type . A
wide range of mutation types including heterozygous one base pair
transitions and transversions and small deletions and insertions could
be detected when compared with wild-type DNA . Our experiments
showed that we were able to obtain different melting curves from wildtype
DNA even for very low level mosaic variants which are detectable
only by allele-specific PCR (AS-PCR) and not by sequencing. A good
sensitivity can be expected when the assay is used to detect otherwise
missed mosaic mutants . HRM analysis is a simple and rapid technique
in prescreening of small sequence alterations in DNA diagnostics .
P08.69
The first serotonin receptor allelic variant database
B. Niesler, R. Röth, S. Wilke, C. Fischer, G. Rapppold;
Institute of Human Genetics, Heidelberg, Germany.
Serotonin (5-hydroxytryptamine, 5-HT) controls a variety of physiological
functions . 5-HT receptors mediating serotonin action are divided
into seven main classes (5-HT 1 R to 5-HT 7 R) . A multitude of candidate
gene screenings has been published during the last years . We have
started to structure this information in the first serotonin receptor allelic
variant database using LOVD (Leiden Open Source Variation Database)
. Up to now, the database comprises data of 5-HT 3 receptor
subunits. To date, five different human subunits are known (5-HT 3A-E ),
which are encoded by the serotonin receptor genes HTR3A, HTR3B,
HTR3C, HTR3D and HTR3E . Different receptor subtypes seem to be
involved in chemotherapy induced nausea and vomiting (CINV), irritable
bowel syndrome (IBS) and psychiatric disorders . During the last
years HTR3 case-control and pharmacogenetic studies indicated that
HTR3A and HTR3B polymorphisms may contribute to the etiology of
psychiatric disorders and may predict CINV and medical treatment of
psychiatric patients. Currently, the database is subdivided into five subdatabases,
referring to the serotonin receptor genes . Within each subdatabase
we are collecting mutations, polymorphisms, demographic
information as well as pharmacogenetic data . Every sub-database
includes general information about the respective gene and is linked
to other resources . The remote user is able to search the data and to
submit new data . This central information pool should help clinicians
as well as scientists to evaluate their findings and to use the information
for subsequent studies . Data about functional consequences of
variants will be integrated in future to enable specific drug design in the
therapy of respective conditions .
P08.70
AutosNP: an integrated graphical user platform of highthroughput
sNP data analysis for disease gene mapping
C. C. Huang 1 , Y. F. Liu 2 , D. D. Lee 3 , C. T. Hsu 4 , Y. T. Chang 3 , T. T. Liu 2 , S. F.
Tsai 5 , M. W. Lin 1,6 ;
1 Institute of BioMedical Informatics, National Yang-Ming University, Taipei, Taiwan,
2 VYM Genome Research Center, National Yang-Ming University, Taipei,
Taiwan, 3 Department of Dermatology, Taipei Veterans General Hospital, Taipei,
Taiwan, 4 Institute of Public Health, National Yang-Ming University, Taipei, Taiwan,
5 Division of Molecular and Genomic Medicine, National Health Research
Institutes, Zhunan Town, Miaoli County, Taiwan, 6 Department of Medical Research
& Education, Taipei Veterans General Hospital, Taipei, Taiwan.
Single nucleotide polymorphisms (SNPs) have become most common
used marker in current human genetic studies . Researchers normally
need to select a set of SNPs from an enormous amount of SNP of candidate
genes or regions . Moreover, current platforms of high-throughput
SNP genotyping will produce huge amounts of genotyping data
with different output format . It is a trivial and time-consuming work to
pick up SNPs and process different kinds of data format for specific
linkage or association analysis program .
To ease the task of managing high-throughput SNPs data from two
main platforms-Illumina and Affymetrix assays, we generated a graphical
user platform named “AutoSNP” by using Perl language . In this
platform, user can select SNP data source and then convert SNP genotyping
data into appropriate format for specific genetic analysis program
. We integrated both linkage analysis programs (Allegro, Gene-
Hunter, Linkage, Merlin, SuperLink and SimWalk2) and association
analysis programs (FBAT, PLINK, HaploView, PHASE, and WHAP)
in this platform . Moreover, this platform also allows users to connect
to Ensembl variation database, NCBI SNP database, and the TAMAL
(Technology And Money Are Limiting) database to query information
from their SNPs list . Finally, we use a skin disease “primary cutaneous
amyloidosis (PCA)” genotyping data as an example to demonstrate
our platform .
P08.71
New methods for sizing Large DNA Fragments on capillary
Electrophoresis instruments
C. Davidson, S. Hung, S. Chen, S. M. Koepf, K. D. Jacobson, M. C. White, S.
Lim, N. Patel, S. Berosik, S. Pistacchi, A. B. Shah, L. K. Joe, R. Santhanam, R.
A. Padilla;
Applied Biosystems, Foster City, CA, United States.
Applied Biosystems is continually developing new fragment analysis
size standards to better support existing and new applications . Recent
developments have focused on improving sizing precision and
accuracy and expansion of the sizing range, using Applied Biosystems’
5th dye technology . We present details about developing methods to
take advantage of a new high performance, 68 peak size standard and
illustrate its versatility, accuracy, flexibility, and precision when used
on various capillary electrophoresis instrument platforms, different
polymers and capillary array lengths . Methods described include DNA
fragment sizing up to 1200bp in applications such as AFLP, T-RFLP,
VNTR, mutation screening, MLST, and BAC fingerprinting.
P08.72
A statistical approach for predicting the clinical type of smA
using mLPA
A. Kastrin, M. Zadel, M. Volk, B. Peterlin;
Division Of Medical Genetics, Ljubljana, Slovenia.
Spinal muscular atrophy (SMA) is a common autosomal recessive disorder
in humans, characterized by degeneration of the anterior horn
cells of the spinal cord, causing symmetric proximal muscle weakness .
SMA is classified in three clinical types, SMA I, SMA II, and SMA III,
based on the severity of the symptoms and the age of onset . The purpose
of this study was to build the prediction model evaluating the
probability of a SMA type based on multiplex ligation-dependent probe
amplification analysis with commercial probe mix (SALSA Probe Mix
021; MRC Holland). This mix contains 16 probes specific for the SMA
critical region (5q12 .2-5q13 .3) . Dosage ratio was chosen to form the
basis of the analysis . We developed a multinomial logistic regression
model based on the data of 65 SMA cases . Prediction model was constructed
using all probes, and using stepwise probe selection algorithm
by Akaike information criterion to remove irrelevant and redundant