European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genomics, technology, bioinformatics
showed that GFP was localized in the same intracellular compartments
as Mito-dsRed. Also HepG2 cells were transfected with this construct.
The cells were collected and subcellular fractions were isolated by differential
centrifugation. Western-blot analysis with antibodies to GFP
revealed GFP in mitochondria. Population comparative analysis of
Cp gene 5’-UTR and translated pCp region in DNA isolated from 117
human blood samples by PCR with following PRLF analysis using of
restriction enzymes set was carried out. The insertions or deletions as
well as mutations in restriction sites in pCp gene amplicons were not
found. At the same time a lot of changes were detected in Cp gene
promoter. These data suggest conservatism of pCP region. In rat, expression
of mitochondrial Cp has tissue specific pattern, not expresses
in newborn and adult animals with copper deficiency, induced by Agfeed.
The biological role of putative protein product of pCp in copper
metabolism is discussed.
P1235. Functional interactions of conserved non-coding
(CNCs) sequences with other CNCs using circular chromosome
conformation capture (4C)
D. Robyr, G. Duriaux-Sail, S. Polti, C. Wyss, S. Deutsch, S. E. Antonarakis;
University of Geneva Medical School, Geneva, Switzerland.
The comparison of human chromosome 21 (Hsa21) with the mouse
syntenic regions led to the identification of roughly 3500 regions displaying
an identity of >70% over a length of a least 100 nucleotides
of ungaped alignment. About 65% (~ 2300) of these are conserved
non-coding sequences (CNCs). Very little is known about the function
of most CNCs. We speculated that a functional CNC would interact
with its genomic target (i.e. an enhancer would bind to it’s cognate
gene promoter). Thus, the identification of any part of the genome that
interacts directly with a CNC could provide clues on the function of the
latter. We have generated libraries of CNC-interacting DpnII fragments
by circular chromosome conformation capture (4C) whose identity is
determined by subsequent sequencing. We have generated initial results
concerning crosslinking of 18 Hsa21 CNCs with DNA fragments
mapping hundreds of kilobases away from the “bait” on the same
chromosome, or with fragments on other chromosomes. A total of 87
such potentially interacting DpnII DNA fragments have been identified.
Interestingly, the median distance from the cloned DpnII fragments to
the nearest conserved region is 661bp with a pvalue < 0.0003 when
compared to the distribution of the median of the distances of 3000
random samples of 87 fragments. These results provide initial evidence
that the function of CNCs is mediated by their interaction with
other conserved regions.
P1236. EM algorithm for gene copy number estimation using
TaqMan® Assays
C. Barbacioru, K. Li, R. Samaha, K. Lazaruk;
Applied Biosystems, Foster City, CA, United States.
Recently, multiple studies have discovered an abundance of copy
number variation of DNA segments ranging from kilobases (kb) to
megabases (Mb) in size. Copy number variations can cause disease,
as in microdeletion or microduplication disorders, or confer risk to complex
disease traits such as HIV-1 infection and glomerulonephritis.
TaqMan® gene copy number assays have been developed for accurate
detection of genetic variation at gene level using primers and
probes designed for genomic DNA. Each well is duplexed with two
assays, a FAM dye-based assay designed to detect the genes-ofinterest
and a VIC® dye-based assay for reference gene. In this study,
we present an algorithm for gene copy number estimation based on
EM algorithm for mixtures of normal distributions. The algorithm finds
maximum likelihood estimates of parameters in probabilistic models,
where the model depends on unobserved samples copy number of
the gene-of-interest. Under current protocols, we are capable of distinguishing
up to 8 copies of the gene of interest with at least 95%
confidence.
To evaluate this algorithm, we present experimental results for 5 important
drug metabolism genes (CYP2D6, CYP2E1, CYP2A6, GSTM1
and GSTT1) on 270 individual samples from International HAPMAP
Project representing 4 different populations. Copy number analysis for
these genes shows perfect consistency for sample duplicates. Copy
number variations are observed for these genes, with significant differences
between these populations. Furthermore, combining this
data with SNP data, we demonstrate that departures from diploidy can
cause apparent genotyping failure and give inaccurate genotyping.
P1237. A computational method to test for genetic relatedness in
unrelated individuals
L. Xumerle, G. Malerba, P. F. Pignatti;
Biology and Genetics, Verona, Italy.
Discovering susceptibility loci in complex disease requires a large
number of individuals characterized by several thousand markers. An
association between gene and disease may be incorrectly estimated
if the allele frequencies differ among cases and controls depending on
factors other than gene-phenotype correlation, i.e. inbreeding, genotyping
errors, unrecognized population stratification, etc.
We present a computational method - available in the Jenoware library
(http://medgen.univr.it/jenoware/) - designed to compute the probability
of genetic relatedness in pairs of individuals. The program computes
the likelihood of a pair of individuals to be unrelated over the likelihood
to be relatives of I, or II degree respectively, conditional to the
genotype. The program works with microsatellites and/or SNPs. False
positive rate and power were assessed by simulation in unrelated individuals
and in pedigrees. E.g. in order to estimate the support for I
degree relatedness (power 80%, and false positive 5%), 10 microsatellites
having heterozygosity >= 70% or 25 SNP having heterozygosity
>= 15% are needed. We are extending the method to associated DNA
markers that can be included as inferred haplotypes in the models to
be tested.
P1238. Genome-wide analysis of copy number variations
in patients with mental retardation by single nucleotide
polymorphism arrays
J. Wagenstaller 1 , S. Spranger 2 , B. Lorenz-Depiereux 1 , B. Kazmierczak 2 , M.
Nathrath 3 , D. Wahl 4 , B. Heye 5 , D. Gläser 6 , V. Liebscher 7 , T. Meitinger 1,5 , T. M.
Strom 1,5 ;
1 GSF - National Research Center, Institute of Human Genetics, Munich, Germany,
2 Praxis für Humangenetik, Bremen, Germany, 3 Technical University,
Department of Pediatrics, Munich, Germany, 4 Praxis für humangenetische Beratung,
Augsburg, Germany, 5 Technical University, Institute of Human Genetics,
Munich, Germany, 6 Zentrum für Humangenetik, Neu-Ulm, Germany, 7 University
of Greifswald, Institute of Mathematics, Munich, Germany.
We investigated 67 children with unexplained mental retardation who
had additional, but often mild symptoms. All children had inconspicuous
high resolution banding analysis. The DNAs were analyzed using
the Affymetrix GeneChip 100K arrays. Data analysis was performed
with median normalization and genotype-specific dosage calculation
using R-scripts and revealed 12 copy number variations (CNVs), 9
deletions and 3 duplications, that are most likely causative for mental
retardation because they either arose de novo or are larger than
known polymorphisms. One of those was a maternally inherited 1.4
Mb duplication in Xp22.31 in a male patient which includes the STS
gene. The chromosome carrying the duplication was non-randomly
inactivated in the mother. Two of the CNVs were de novo deletions
affecting only a single gene. All CNVs were confirmed by quantitative
PCR. They varied in size from 200 kb to 8 Mb. Five of the CNVs were
flanked by low-copy number repeats. Three of them were known microdeletion
syndromes. The fourth one is a deletion on chromosome
15q25.2 that was not described before. The fifth one is the duplication
in Xp22.31. We compared different array types. The signal-to-noise
ratio (SNR) of the 500K Affymetrix array was lower than the SNR of
the 100K Affymetrix array. The SNR of the 300K Illumina arrays was
in between. The increase of the number of features and a new design
should improve the resolution and allow the reliable detection of gain
and losses of single genes.
P1239. Multiplex Amplicon Quantification, a novel method for
PCR based, high-throughput copy number variation analysis.
D. Goossens 1,2 , P. De Rijk 1,2 , L. Heyrman 1,2 , B. Harding 3,2 , W. Glassee 3,2 , J.
Del-Favero 1,2 ;
1 Applied Molecular Genomics Group, Department of Molecular Genetics, VIB,
Antwerp, Belgium, 2 University of Antwerp, Antwerp, Belgium, 3 Department of
Molecular Genetics, VIB, Antwerp, Belgium.
Recently, a striking abundance of copy number variation (CNV) was
discovered throughout the human genome. Although it has been sug-
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