2008 Barcelona - European Society of Human Genetics

Genetic counselling, education, genetic services, and public policy
Presently, the usefulness of WGA in STR-based forensic analysis is
limited because of allelic dropout (ADO) and bias in peak area ratios
observed in low copy number (LCN) templates . Sequence polymorphisms
(SNPs) are more sensible than length-polymorphisms (STRs)
and less prone to ADO (allelic dropout) when WGA is applied . Thus,
we recently validated a panel of TaqMan SNP assays selected to show
an high sensibility in LCN templates . Here we evaluated the performance
of multiple displacement amplification (MDA) applied to our
optimized SNP assays starting from small amounts of genomic DNA
(gDNA) . A set of 100 samples were analyzed for 21 SNPs and a total of
1 ng, 100 pg and 10 pg of genomic DNA of each sample was used as
template for the MDA. Concordance and amplification failure (AF) between
gDNA and wgaDNA were extremely robust (100%) when WGA
was performed on 1 ng and 100 pg of gDNA, whilst the concordance
decreased to 99.2% for samples amplified from 10 pg of gDNA. The
absence of a full concordance for 10 pg samples is referred as ADO
occurring when a gDNA heterozygote genotype is scored as homozygote
but it should not lead to mis-typing if only heterozygous genotypes
are considered. The robustness of WGA applied to specifically
selected SNP assays should suggest a reconsideration of WGA for
forensic SNP profiling.
P08.86
comparison of genotyping consistency between genomic and
whole-genome amplified DNA using the Illumina GoldenGate
and Infinium-II assays
S. Cichon1,2 , M. Alblas1 , K. Kämmerling1 , T. W. Mühleisen1 , M. M. Nöthen1,2 , P.
Hoffmann1,2 ;
1 2 Life & Brain Center, University of Bonn, Bonn, Germany, Institute of Human
Genetics, University of Bonn, Bonn, Germany.
High-throughput SNP genotyping has become an important research
strategy in human genetics . Although most genotyping assays require
minimal amounts of DNA, repeated use often leads to depletion of
the samples. To address this problem whole-genome-amplification
(wga) technologies have been developed and are meanwhile commercially
available. Albeit the amplification seems to be mostly successful,
it is controversially discussed whether the wgaDNA represents
an exact copy of the genomic DNA (gDNA) . In the present study, we
aimed to assess the genotyping consistency between 45 wgaDNAs
(generated using the REPLI-g DNA Amplification Kit, Qiagen, Hilden)
and their corresponding gDNA samples . The gDNAs were of different
age and quality . To compare genotype consistency 20 high-quality
sample pairs were genotyped using Illumina’s HumanHap550V3
BeadChips .(565 .000 SNPs) . 25 sample pairs of different DNA quality
were genotyped for 384 SNPs using Illumina’s GoldenGate assay . All
samples genotyped on the HumanHap550V3 performed well, with call
rates >99% . The average consistency between gDNA and wgaDNA
was 99 .99% when comparing SNPs successfully genotyped in the corresponding
samples . Of the 25 sample pairs genotyped with the GoldenGate
assay, 22 performed well with call rates >99% (gDNA) and
>98% (wgaDNA) . Genotype consistency was 100% for corresponding
samples . The remaining 3 sample pairs showed noticeably worse results
with an average genotype call frequency of 99 .8% (gDNA) versus
60 .1% (wgaDNA) and a genotype consistency of only 89% . Possible
explanations for the observed discrepancies include the age of gDNA,
the extraction method as well as the presence of unknown inhibitors
interfering with the amplification process.
P08.87
comprehensive Desktop software for Next Generation
sequencing Applications
S. Baldwin, D. Nash, K. Dullea, R. Nelson, T. Durfee, J. Engelking, F. Blattner;
DNASTAR Inc., Madison, WI, United States.
Next Generation sequencing technologies provide the data generation
tools for many large scale molecular biology applications including
whole genome sequencing/resequencing, polymorphism detection,
as well as gene expression and regulation . The cost effectiveness of
these technologies makes them accessible to virtually any researcher .
One of the lagging issues, however, has to do with the handling of
the large quantities of data generated and gaining access to the tools
required to analyze the data . To provide users with the ability to take
advantage of the next-gen revolution, DNASTAR has developed fully
scalable software capable of processing a wide range of resequenc-
ing and whole genome projects on a desktop computer . The software
is fully compatible with Sanger, Roche 454, Illumina and ABI SOLiD
sequence platforms. We will provide workflow examples of assembly
within SeqMan Genome Assembler for rapid resequencing assembly
projects along with the use of its companion, SeqMan Pro for finishing,
analyzing and annotating whole genome assemblies . We will also
present a workflow for using the software in digital gene expression
applications comparing data from microarrays to data generated by
next generation sequencing instruments .
P09. Genetic counselling, education,
genetic services, and public policy
P09.01
Diagnostic and clinical validations in DNA-diagnostic
laboratories: a BRcA example
D. Bodmer, M. R. Arjen, S. Hans, H. Nicoline, L. J. L. Marjolijn;
Department of Human Genetics, Nijmegen, The Netherlands.
Clinical validations are used to assess the clinical sensitivity and specificity
of genetic testing within DNA diagnostic laboratories and are an
important instrument to monitor and further improve reliability and efficiency
of molecular testing. However, for most indications the clinical
information available to DNA diagnostic laboratories is too scarce
to determine the a priori chance of a positive test result . In order to
assess the sensitivity and specificity of genetic testing, we started
with a so called diagnostic validation, in which the robustness of the
method(s) to analyze a single gene or a set of genes for a genetic disease
was determined . The parameter used is the mutation detection
ratio (MDR) which is defined as the proportion of mutation-positive results
. By comparing the MDR of a genetic test at different time intervals
(e .g . years), the robustness of this test can be judged . This robustness
depends on changes and variations that occur within a method (with
or without preceding analytical validation) as well as method performance
by different operators . Decrease of the MDR of a genetic test
over time, should lead to critical evaluation of the different laboratory
processes and assessment of putative alterations in the a priori risk of
the diagnostic requests .
An example: The efficiency of BRCA1 and BRCA2 mutation analysis
has decreased within years . To determine the cause of this decrease,
e .g . whether this is due to technical issues or solely to an altered referral
policy, we performed both a diagnostic validation as well as a
clinical validation .
P09.02
Expanded newborn screening: challenges for the provision of
pre-newborn screening care
J. Allanson 1 , F. A. Miller 2 , R. Hayeems 2 , J. Carroll 3 , P. Chakraborty 1 , R. Christensen
2 , J. Little 4 , B. Wilson 4 ;
1 Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada, 2 University of
Toronto, Toronto, ON, Canada, 3 Mount Sinai Hospital, Toronto, ON, Canada,
4 University of Ottawa, Ottawa, ON, Canada.
Historically, newborn screening (NBS) aimed to identify serious childhood
disorders for which treatment was available to reduce morbidity
and mortality . Limited attention to pre-screening information provision
was justified by the severity and treatability of the conditions screened.
Yet technological advances allow NBS programs to identify disorders
for which the promise of clinical benefit is uncertain as well as an array
of “incidental” findings (benign variants, carrier status results). Given
the contested value of such results, commentators argue that limited
attention to pre-screening care is no longer justifiable. This paper reports
survey data on attitudes and practices of a cross-sectional stratified
random sample of five health care professional (HCP) groups in
Ontario that are involved in prenatal care and/or care of newborns in
the first days of life (obstetricians, midwives, nurses, family physicians,
pediatricians) . The majority of HCPs surveyed (68%) believe it is their
responsibility to provide information about NBS to parents prior to the
heel prick test . However, as many as 48% of these providers report
that they do not consistently or usually do so . This paper explores the
role of provider type, practice barriers (e.g. insufficient time, training,
compensation) as well as knowledge and confidence of NBS in explaining
the discrepancy between perceived professional responsibility
and actual professional practice . Thus, while most HCPs perceive