1. KCNE1 SNP fine mapping of Gene and flanking sequences
Functional Genomics by Association Study (Lesson from KORA Study)
Shapour Jalilzadeh1,3; Stefan Kä ä b2
; Bianca Hildebrand2; Mahmut Akyol3;Ludwig Zwermann2;
Michael Nä bauer2
; Thomas Meitinger1,3
; Arne Pfeufer1,3
1. Institute for Human Genetics, Technical University of Munich, Germany. 2. Department of Medicine I, Klinikum Grosshadern, LMU-University, Munich, Germany
3. Institute for Human Genetics, GSF National Research Center, Neuherberg, Germany
Material and Methods: 48 explant heart tissues (Left ventricle) were
analyzed for different splice variants for KCNE1. cDNA ends were amplified and
sequenced with primers specific to different splice variants. Splice variants were
quantitated with Taqman probes. Exon 5 was used as control because it is
present in all splice variants. All experiments were performed in dublicates.
Averages values were taken. In parallel SNPs rs727957 and rs1805127(G38S)
was genotyped on DNA extraced from the same cardiac tissues by Taqman
probes and MALDI-TOF Mass Spectrometery.
Introduction: Much of the complexity of higher organisms is attributable to evolution of regulatory elements rather than exons. Alternative promoters are common to at least half of
all human genes. The function of regulatory elements can be modulated by allelic variants (regulatory SNPs), which may affect transcription levels, alternative splicing and mRNA
stability as well as translation or post-translational modification.1
In a candidate gene based LD-mapping we discovered a SNP (rs727957 G>T) located 50 kb upstream of the KCNE1 gene (Fig 1) which had an effect on QT interval in the general
population (+1.2 ms/allele in KORA S4, p =0.0051)2
. Bioinformatic analysis shows the transversion can produce a new transcription factor binding site for the muscular transcription
factor TEF-1 which is expressed in heart tissue. Analysis of cDNAs from 48 different human hearts show that 5 exons of KCNE1 exist, where only exon 5 contains the entire ORF. At
least 6 splice variants and 3 alternative promoters of KCNE1 exist but identified transcripts are translated to the same protein. SNP such as rs727957 may influences alternative
promoter usage of KCNE1. HapMAp data (Fig 2) indicates this SNP may be under selection (polymorphic in CEU but monomorphic in HCB, JPT and YRI populations).
Figure 1. SNP rs727957 is located 50 kb upstream of the KCNE1 gene
which can modulate QT interval in the general population KORA S4.
Figure 2.HapMap data suggest selection force on SNP rs727957 because of being
polymorphic in CEU(MAF 18%) and not other population (HCB, JPT and YRI ).
Result: We detected 6 different splice variants in human LV heart tissue, Relative expression levels of which differt inter individually.
Figure.5. Splice variants were quantitated with Taqman specific probe.
As it is appear each variants expressed differentially in heart tissue.
Our findings suggest there are at least 3 alternative promoters for KCNE1 located in front of exon1, exon 3 and exon 5. Activation or silencing of promoters in variable situations can
explain effect of KCNE1 up- or down-regulation on QT interval in normal and disease states. Genotyping data of rs1805127(G38S) showed a weak association with level of splice
variants(p<0.1). We are going to analysze 100 heart tissue samples to achieve sufficient statistical power for reliable analysis..
Conclusion:
.Non-genic DNA gets more Importance such as CNG(conserved non-genic
DNA) and LCRs.
.Linkage and association study (especially SNP fine mapping) can help to
find, map and functional analysis of regulatory SNPs,ie understood.
References:
1-Prokunina L, Alarcn-Riquelme ME. Regulatory SNPs in complex
diseases: Expert Rev Mol Med. 2004 Apr 27;2004:1-15
2-Pfeufer A, Jalilzadeh S, Perz S, Mueller JC, Hinterseer M, Illig T,
Akyol M, Nabauer M, Wichmann HE Meitinger T, Kaab S. Common
variants in myocardial ion channel genes modify the QT interval in
the general population.Circ Res. 2005 Apr 1;96(6):693-701. Epub
2005 Mar 3.
Figure .4.At least 6 different splice variants of KCNE1exist in LV tissue. Two
kinds of exon 4(short and long) differ in 27 nucleotides.
Figure 3.Exon-Exon Junction strategy is efficient method to capture
and quantitate splice variants
Variant 1
Variant 2
Variant 3
Variant 4
Variant 5
Variant 6
Ex5 Ex4l Ex2 Ex1
Ex5 Ex4s Ex2 Ex1
Ex5 Ex2 Ex1
Ex5 Ex4l Ex3
Ex5 Ex4s Ex3
Ex5