2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics
P08.81
the Human Variome Project - plans and progress
R. G. H. Cotton1,2 ;
1 2 Genomic Disorders Research Centre, Carlton South VIC, Australia, Convenor,
The Human Variome Project, Australia.
The Human Variome Project (HVP; www .humanvariomeproject .org)
was created to coordinate and curate the collection of all genetic variation,
its phenotype and associated disease(s) . This is because lack
of up-to-date, complete and correctly curated information can lead to
excessive web searching, misdiagnosis and wastes valuable healthcare
funds .
Work to obviate this problem began more than a decade ago with the
Human Genome Variation Society (HGVS; www .hgvs .org) promoting
collection and display of variants, producing recommendations and
software .
At the launch of the HVP, 96 recommendations (www .nature .com/ng/
journal/v39/n4/pdf/ng2024 .pdf) were drawn up by over 50 world experts
from over 20 countries to be implemented in the future . The task
is large so countless people will need to be involved in a coordinated
manner with specially developed tools and protocols .
What is needed is an automated, seamless system transferring clinical
data (phenotype), genotype and pathological data to hospital records,
as well as to databases curated by experts, in a de-identified
and ethically acceptable way, initially to LSDBs and finally to central
databases/browsers .
The International Society for Gastrointestinal Hereditary Tumours (In-
SiGHT; www .insight-group .org) has volunteered to be a pilot for (a)
collection of all mutations and phenotype for their four genes of interest
and (b) from all countries .
Many components for this flow have already been developed, often
multiple times around the world in an un-coordinated disconnected
way . A planning meeting was held in May 25-29 2008 to review
these and rationalise future planning (www .humanvariomeproject .org/
HVP2008/) .
P08.82
teststrip-based genotyping to assist in the prediction of
anticoagulant dose requirement
H. Puehringer 1 , Q. Berisha 2 , G. Klose 3 , B. Schreyer 3 , W. Krugluger 2 , R. Loreth
3 , C. Oberkanins 1 ;
1 ViennaLab Diagnostics GmbH, Vienna, Austria, 2 Department of Clinical Chemistry,
Rudolfstiftung Hospital, Vienna, Austria, 3 Clinical Haemostaseology, Westpfalz-Klinikum
GmbH, Kaiserslautern, Germany.
Coumarin derivatives, such as warfarin and phenprocoumon, are
the most widespread oral anticoagulant drugs for the prevention and
treatment of arterial and venous thromboembolic disorders . However,
these vitamin K antagonists have a narrow therapeutic range and a
wide interindividual variability in dose requirement . Despite adjustment
for clinical variables, adverse events are frequently encountered during
the initial phase of therapy . Genetic polymorphisms in the drugtargeted
vitamin K epoxide reductase complex 1 (VKORC1) and in the
drug metabolizing enzyme CYP2C9 have been reported to account for
the majority of variations in the therapeutic response to warfarin .
We have developed a genetic test (StripAssay) for the simultaneous
detection of two VKORC1 polymorphisms (-1639G>A, 3730G>A) and
the functionally defective CYP2C9 variants *2 and *3 determined by
430C>T and 1075A>C . Preliminary data from our ongoing clinical
study, to date including 130 patients treated with phenprocoumon
(Marcumar), allowed a classification of high, intermediate and low
dose responders according to VKORC1 and CYP2C9 genotypes . The
stable dosage required for therapeutic anticoagulation was considerably
lower in carriers of a combined VKORC1 -1639A and CYP2C9 *2
or *3 genotype compared to carriers of a single variation or wildtype
alleles . The VKORC1 3730G>A polymorphism seemed to have no additional
predictive power for phenprocoumon dose variability .
The new diagnostic assay and the results obtained during our study
will assist clinicians to achieve a safer and more individualized anticoagulant
therapy .
P08.83
eSensor® genotyping test for CYP2C9, CYP4F2 and VKORC1
polymorphisms associated with warfarin sensitivity
W. A. Coty, M. R. Reed, A. R. Jacobs, P. Naranatt, R. Hubert, S. Panuganti, Z.
Wang, V. Headley, Y. Liu, M. Abedi, G. R. Gust, K. Olszewski, D. Canfield;
Osmetech Molecular Diagnostics, Pasadena, CA, United States.
We have developed an eSensor® test to genotype 8 CYP450 2C9
polymorphisms known to affect enzyme activity (2C9 *2, *3, *5, *6, *11,
*14, *15 and *16), as well as the VKORC1 -1639G>A promoter polymorphism
associated with warfarin sensitivity and the CYP450 4F2
rs2108622 (V433M) polymorphism recently found to correlate with
warfarin dose . After multiplex PCR and exonuclease digestion, genotyping
is performed using the eSensor® cartridge and XT-8 instrument
within 35 minutes for up to 24 samples . In a reproducibility study performed
with 20 genomic DNA samples and 3 plasmid controls (n = 345
tests), 100% first-pass call rate and agreement with DNA sequencing
were obtained . A method comparison study with 105 genomic DNA
samples extracted from blood gave 100% call rate and agreement with
DNA sequencing after re-testing of no-call samples, as did an additional
cohort of 145 genomic DNA samples from saliva and cell lines .
The assay gave 100% first-pass call rate and agreement with DNA
sequencing using input genomic DNA amounts between 10 and 1,000
ng per PCR . Testing of ethnic panels from the Coriell Cell Repository
revealed elevated allele frequencies for 2C9 *5 (3%) and *11 (7 .6%)
in the African-American panel (n=33), and 2C9 *14 (2 .2%) in the Gujarathi
Indian panel (n=90) . Initial feasibility has been demonstrated
for PCR amplification directly from whole blood samples, with no interference
observed from serum albumin, IgG, bilirubin, hemoglobin,
triglycerides or excess EDTA .
P08.84
A multiplex detection assay for Warfarin dosing using single
Base Extension
A. J. Rai1 , N. Udar2 , C. T. Yu1 , M. Fleischer1 ;
1 2 Memorial Sloan Kettering Cancer Center, New York, NY, United States, Beckman
Coulter, Fullerton, CA, United States.
Thromboembolic events in “at risk’ individuals can be prevented by anti
coagulation drug therapy . Warfarin is a commonly used anticoagulant
prescribed to over one million patients in the US (2006) . Individuals
vary greatly in their response to warfarin therapy . This difference has a
genetic basis . Two genes: cytochrome P450 2C9 (2C9) and vitamin K
epoxide reductase subunit protein 1 (VKORC1) have been reported to
account for 60% of these differences . There are two clinically important
alleles of 2C9(*2 and *3) and one of VKORC1 (-1639 G->A) . We designed
a multiplex assay for the simultaneous detection of these three
alleles in a single reaction. Our assay entails a multiplex PCR amplification
of the target gene fragments followed by a multiplex single base
extension reaction . The single base extension reaction incorporates the
target nucleotide which has a fluorescent tag attached to it. This tag
is detected by separation on a capillary electrophoresis platform . The
entire assay can be performed with in an eight hour day by a single technologist
with minimal hands-on effort . We observe 100%concordance
on twenty samples when our assay is compared to traditional DNA sequencing
. We have optimized this assay for high-throughput screening
of patient samples, allowing for analysis of two 96well plates on a single
overnight run . Our multiplex SNP panel can be used as a stand-alone
test for patients starting warfarin therapy, or its results can be combined
in an algorithm with additional parameters (e .g . weight, age, sex, etc .) to
provide dosing recommendations for initial warfarin administration .
P08.85
Whole Genome Amplification (WGA) in forensic SNP profiling
I. Pietrangeli 1 , C. Martone 1 , E. Giardina 1 , I. m. Predazzi 1 , P. Marsala 2 , l. gabriele
3 , c. pipolo 3 , o. ricci 3 , G. Solla 3 , A. Spinella 3 , G. Novelli 1,4 ;
1 Centre of Excellence for Genomic Risk Assessment in Multifactorial and Complex
Diseases, School of Medicine, Tor Vergata University of Rome, Rome,
Italy, 2 Direzione Centrale Anticrimine, Servizio di Polizia Scientifica, Rome, Italy,
3 Direzione Centrale Anticrimine, Servizio di Polizia Scientifica, Rome, Italy,
Rome, Italy, 4 Division of Cardiovascular Medicine, Department of Medicine,
University of Arkansas for Medical Sciences, Little Rock, AR, United States.
Whole genome amplification (WGA) is a technique developed for genetic
analysis to obtain sufficient amount of DNA from small pools of
cells or even single cells .