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Molecular characterization of the DYX1C1 gene and its
application as a cancer biomarker
Heui-Soo Kim1, Yun-Ji Kim1, Jae-Won Huh1,2, Dae-Soo Kim1,3, Yi-Deun Jung1, Hong-Seok Ha1, and Won-Ho Lee1
1 Division
of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea
2 National Primate Research Center (NPRC), KRIBB, Ochang, Chungbuk 363-883, Republic of Korea
3 PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea
Heui-Soo Kim e-mail: [email protected], Yun-Ji Kim e-mail: [email protected]
http://www.primate.or.kr
Abstract
DYX1C1 has three alternatively spliced transcripts. So we expect that alternative transcripts of DYX1C1 are used as a biomarker to detect specific
cancer. RT-PCR analysis is conducted in order to detect expression of the DYX1C1 gene and the PCR products were analyzed using the Image J
program to compare the expression levels of each transcript. We found one of the transcripts was directly associated with an HERV-H LTR
element that could be translated into protein sequence. Four new alternative transcripts were identified by RT-PCR analysis with various
human tissue samples including 5 normal and adjacent tumor tissue sets. Semi-quantitative RT-PCR analysis showed the transcriptional activity
of V3 and V2 was higher in normal than in tumor tissue samples, especially in the colorectal tissue samples. Our results indicated that
alternatively spliced transcript variants of the DYX1C1 gene could be used as cancer biomarkers to detect colorectal cancer.
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Introduction
Materials and Methods
Biomarker
The identification of cancer biomarkers has been a major focus of investigation in the field of
cancer research. However, effective and reproducible cancer biomarker developments are still far off
and enormous efforts are needed in this area (Brinkman et al. 2004). In cancerous cells, splicing
mechanisms are significantly altered by defects in splice sites caused by frequent point mutations
(Venables et al. 2004; Skotheim et al. 2007). Aberrant neuronal-specific splicing of amphiphysin 2 in
non-neuronal cells may contribute to the progression human melanoma (Ge et al. 1999). Expression
levels of alternatively spliced variants of survivin are not equal to each other in malignant breast
tissues. In the case of breast cancer, different survivin transcripts show different functions in the
progression of apoptosis (Ryan et al. 2005; Pajares et al. 2007). Our study focused on the
application of cancer biomarkers using transcription variants.
Bioinformatics
( in silico analysis DYX1C1 gene )
PCR and RT – PCR in human tissues
and various normal/tumor sets
Alignment & Image J analysis
Application to colorectal patients
DYX1C1
DNA
DYX1C1 is a recently identified candidate gene for dyslexia. Disruption of the gene by a
translocation was detected in dyslexia patients (Taipale et al. 2003; McGrath et al. 2006). The
DYX1C1 gene maps to chromosome 15q21 and consists of 10 exons dispersed over about 78 kb of
genomic DNA. The protein sequence encoded by DYX1C1 is 420 aa in length and the protein
sequences of the nonhuman primate homolog are 98.6-99.5% similar to that of humans (Taipale et
al. 2003). DYX1C1 is expressed broadly in several adult tissues including lung, kidney, and brain
(Taipale et al. 2003; Wang et al. 2006), where its protein is localized in white matter including the
nuclei of cortical neurons and glial cells (Taipale et al. 2003; Fisher and Francks 2006). Eight single
nucleotide polymorphisms (SNPs) in the DYXICI genes of 20 patients with reading disabilities were
detected (Taipale et al. 2003; McGrath et al. 2006). The 1249G-to-T and -3G-to-A mutations were
among the SNPs found more frequently in patients with dyslexia than in controls (Fisher and Francks
2006). Developmental dyslexia is associated with migration anomalies in the neocortex as well
genetic susceptibility (Pennington and Smith 1983; Galaburda et al. 1985; Smith et al. 1998; Chang
et al. 2005; Sokol et al. 2006). DYX1C1 plays a role in the migration of neocortical neurons.
Specifically it is required for the transition of the multipolar stage of migration (Wang et al. 2006).
Human genomic
DNA
vector
PCR, RT-PCR
Electrophoresis
ligation
Bioinformatics
Sequencing
analysis
Plasmid isolation
inoculation
transformation
Results and Discussion
AS1
S1
EXON1
2
3
4
5
6
7
8
9
V1
NM_001033560.1
EXON1
2
3
4
5
6
7
8
8’
9
NEW TRANSCRIPTS
V1-1
EXON1
2
3
4
5
6
7
8
8’
9
V1-2
EXON1
2
3
4
5
6
7’
7
Fig.4 Comparative analysis of the other transcripts (V2, V3)
in normal (N) and tumor (T) samples. G3PDH indicates the
positive control
9
V1-3
EXON1
2
3
4
5
6
7
9
V1-4
AS2
S2
EXON1
2
3
4
5
6
7
8
Fig.2 Expression patterns of HERV-H LTR-associated (A) and unassociated (B) DYX1C1 transcripts in normal human tissues.
G3PDH indicates the positive control.
9
V2
NM_001033559.1
AS2
S2
EXON1
2
3
4
5
6
7
8
9
10
NM_13081.2
V3
Fig. 3,4 - (b) RT-PCR was conducted three times and G3PDH
was used as a control. PCR products were analyzed
quantitatively using the Image-J program. The X-axis of the bar
graph indicates normal (N)/tumor (T) tissue samples. Lanes: M,
size marker; 1, colon (N); 2, colon (T); 3, liver (N); 4, liver (T); 5,
uterus (N); 6, uterus (T); 7, breast (N); 8, breast (T); 9, stomach
(N); 10, stomach (C) and the Y-axis of the bar graph indicates
the relative expression levels of 2 transcripts (V2 and V3). P <
0.05 inStudent’s t-test is indicated by *.
Fig.1 Spliced variants of the DYX1C1 gene and
their structural analysis
The DYX1C1 gene is shown to have three alternatively
spliced transcripts in the GenBank database. V1, V2, and V3
are the originally reported transcripts, and V1-1, V1-2, V1-3,
and V1-4 are newly identified alternative transcripts.
Fig.3 Comparative analysis of HERV-H LTR-associated DYX1C1
transcripts (V1, V1-1, V1-2, V1-3, V1-4) in normal and tumor tissue
samples. G3PDH indicates the positive control.
Fig.5 Comparative analysis of the other transcripts (V2, V3)
in normal (N) and tumor (T) samples of colorectal cancer
patients. G3PDH indicates the positive control
References
1. Adran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, Inuzuka M. (2004) Identification of a novel splice variant of the human anti-apoptopsis gene survivin. Biochem Biophys Res
Commun. 314: 902-907.
2. Brinkman BM. (2004) Splice variants as cancer biomarkers. Clin Biochem. 37: 584-594.