2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
melt only.
Ten SNPs were interrogated using each method. Comparisons included
genotype accuracy, discrimination of unequal allele fractions,
and simplicity of design. SAG assays used primers placed as close as
practical to each SNP, generating 50-70 bp amplicons. LunaProbes
and Snapback utilized the same primers, generated 90-150 bp amplicons,
and were designed to work using asymmetric PCR. LunaProbes
were designed 18-35 bp. Snapback used one primer that was modified
with a 5’ probe element.
Each method was 100% accurate in genotype calls across all targets,
with all genotypes represented. SAG assays were easiest to design
and optimize. LunaProbes and Snapback required the lowest software
sensitivity setting to call accurate genotypes, and displayed greater discrimination
of unequal alleles, down to 5% of the low-fraction allele.
All three applications are accurate, cost-effective and offer different
advantages. Together, these methods will offer increased flexibility and
success rate of assay development. SAG is the simplest method, while
LunaProbes and Snapback offer greater ability to distinguish unequal
allele fractions.
P11.056
A PcR coupled high-resolution melting analysis for reliable gene
scanning of the faciogenital dysplasia gene, FGD1
T. Kaname 1,2 , K. Yanagi 1 , Y. Chinen 1 , K. Naritomi 1,2 ;
1 University of the Ryukyus, Nishihara-cho, Japan, 2 SORST, JST, Tokyo, Japan.
It is important to establish an easy and reliable system to detect mutations
or variations for genetic testing. High-resolution melting analysis
(HRM analysis) is a method, which allows simple and rapid detection
of gene variations. We constructed a sensitive system for detecting
gene variations in FGD1. The FGD1 gene is a responsible gene for
Aarskog-Scott syndrome (AAS), which is an X-linked disorder characterized
by short stature, dysmorphic facial appearance, brachydactyly,
shawl scrotum, and sometimes neurobehavioral impairment.
We set up a PCR coupled HRM system for all exons of FGD1 using
LightCycler 480 Instrument (Roche). Then we evaluated the PCR/
HRM in the screening seven mutations of FGD1, which we found in
AAS patients previously, plus variations of FGD1 in five sporadic patients,
two families, and 48 controls. The PCR/HRM discriminated all
the FGD1 mutations studied from wild-type DNA. In control individuals,
four polymorphisms and three unknown variations were found in the
FGD1 gene. Besides, the PCR/HRM discriminated not only four haplotypes
in exon 14, but also between heterozygous and hemizygous or
homozygous of those haplotypes.
The system is a valuable method for rapid and reliable scanning of
FGD1 gene variations and is applicable to high-throughput genetic
testing.
P11.057
Functional study of nuclear missense mutations causing
mitochondrial HMG-CoA synthase deficiency
M. Ramos, M. Arnedo, B. Puisac, M. C. Gil-Rodríguez, M. P. Ribate, J. C. de
Karam, A. L. Díaz, S. Menao, F. J. Ramos, J. Pié;
Laboratory of Clinical Genetics and Functional Genomics. Medicine School.,
Zaragoza, Spain.
The HMGCS2 gene codifies the human mitochondrial HMG-CoA
synthase (mHS). The mitochondrial HMG-CoA synthase deficiency
(OMIM 600234) is a rare autosomic recessive disorder of the ketonebodies
synthesis that sometimes cause sudden infant death. Up to
date, eight patients with this deficiency have been reported. Clinical
diagnosis must be confirmed by measuring the enzyme activity in liver
tissue. Recently, we have cloned the HMGCS2 gene into the pMALc2x
expression vector, overexpressed the recombinant plasmid into
the E.coli BL21 and purified the mHS protein with amilose affinity chromatography.
Here, we reported for the first time the obtention of the
mHS protein correctly folded and with a great level of purity which
allowed us to carry out accurate enzyme activity measurements. We
have also studied the natural mutants of the enzyme and observed
significant structural abnormalities in four of them. We identified two
missense mutations that caused the complete loss of the enzyme activity
and one missense mutation that caused the loss of 75% of enzyme
activity.
This work is supported by a grant of Diputación General de Aragón
(Ref. B20).
P11.058
Genomic targets of human papillomavirus E2 protein
R. Kurg1 , L. Võsa1 , A. Sudakov2 , M. Ustav1 , M. Remm2 ;
1 2 Institute of Technology, Tartu, Estonia, Institute of Molecular and Cellular Biology,
Tartu, Estonia.
Papillomaviruses are DNA tumour viruses that infect epithelial cells
and induce the formation of benign hyperproliferative lesions. HPV E2
protein regulates viral gene transcription and is required for viral DNA
replication. E2 is a sequence-specific DNA-binding protein that recognizes
a palindromic sequence 5’-ACCGNNNNCGGT-3’ (E2BS). Our
aim was to study the involvement of papillomavirus E2 protein in regulation
of cellular gene expression. We have identified the abundance
and placement of E2BS in the human genome and studied the role of
some of these sequences in E2-dependent cellular transcription regulation.
Bioinformatics analysis revealed that human genome contains
over three thousand copies of E2-specific DNA motifs, but only 753 of
them are locating in repeat free regions. E2BSs occur less frequently
than is expected from genome nucleotide content. Additionally, most
sites are suboptimal for HPV E2 binding suggesting that the number
and structure of these sites appears to be under negative selection
pressure. Our experiments show that some of the genomic sequences
containing at least two E2BS within 500 bp can act as E2-responsive
transcription regulatory elements in transient transcription assays.
E2 can also induce changes in expression levels of genes containing
those sites in the genomic context. These data suggest that HPV E2
proteins can alter cellular gene expression through binding to specific
E2 recognition sites in the human genome.
P11.059
Primer optimization for detecting low levels of methylation using
High-Resolution melting analysis
A. R. Tobler, N. Koch, G. Janaway, C. J. Davidson, M. J. O’Donoghue;
Applied Biosystems, Foster City, CA, United States.
DNA methylation plays a critical role in the regulation of gene expression
in development, differentiation, and disease. Methylation of CpG
Islands in promoter regions usually turns off gene transcription. Global
hypomethylation of genomic DNA has been observed in tumor cells
and a correlation between hypomethylation and increased gene expression
has been reported for many oncogenes.
Most of the techniques available to study DNA methylation do not offer
enough sensitivity to confidently detect methylated DNA levels less
than 10%. HRM is a powerful method, able to achieve detection levels
below 10%; however, knowledge of different parameters that influence
detection sensitivity will improve the chance of a successful assay.
In this report, methylation of the promoter region of the MTA1 gene is
examined using HRM. Positive controls with 100% and 0% methylation
are used to establish melt curves for complete hyper- and hypomethylation,
respectively. Unknown samples with as low as 0.1% methylation
are detected using the HRM method. This can be achieved by
including CpG dinucleotides in the PCR primer sequence and deliberately
shifting the PCR bias to preferentially amplify the methylated
sequence. The methylation status of each CpG is confirmed by directly
sequencing the PCR product of the HRM reaction. These results
demonstrate the sensitivity of HRM analysis in detecting extremely low
levels of methylation, and the general workflow of sequencing the PCR
product following HRM methylation analysis.
P11.060
Rapid high throughput screening and identification of unknown
DNA variation using High-Resolution melt and sequencing
workflow.
N. Koch, N. Chen, A. Lam, G. Janaway, J. Wang;
Applied Biosystems, Foster City, CA, United States.
High Resolution Melt (HRM) is a recent enhancement to traditional
melting analyses that significantly increases the detail and information
that can be captured. HRM requires the use of new DNA binding
dyes that allow a sharp melting transition of double stranded to
single stranded DNA. The HRM screening method is sensitive enough
to detect single nucleotide differences in several hundred base pairs
of sequence. We have successfully used HRM to screen for unknown
DNA variations in genomic DNA. Traditional methods of DNA variant
screening (dHPLC, SSCP) are more costly and time consuming
than the HRM method. HRM screening identifies samples containing