- Research article
- Open Access
Paternal imprinting of the SLC22A1LS gene located in the human chromosome segment 11p15.5
© Bajaj et al; licensee BioMed Central Ltd. 2004
- Received: 05 February 2004
- Accepted: 03 June 2004
- Published: 03 June 2004
Genomic imprinting is an epigenetic chromosomal modification in the gametes or zygotes that results in a non-random monoallelic expression of specific autosomal genes depending upon their parent of origin. Approximately 44 human genes have been reported to be imprinted. A majority of them are clustered, including some on chromosome segment 11p15.5. We report here the imprinting status of the SLC22A1LS gene from the human chromosome segment 11p15.5
In order to test for allele specific expression patterns, PCR primer sets from the SLC22A1LS gene were used to look for heterozygosity in DNA samples from 17 spontaneous abortuses using PCR-SSCP and DNA sequence analyses. cDNA samples from different tissues of spontaneous abortuses showing heterozygosity were subjected to PCR-SSCP analysis to determine the allele specific expression pattern. PCR-SSCP analysis revealed heterozygosity in two of the 17 abortuses examined. DNA sequence analysis showed that the heterozygosity is caused by a G>A change at nucleotide position 473 (c.473G>A) in exon 4 of the SLC22A1LS gene. PCR-SSCP analysis suggested that this gene is paternally imprinted in five fetal tissues examined.
This study reports the imprinting status of the SLC22A1LS gene for the first time. The results suggest imprinting of the paternal allele of this gene in five fetal tissues: brain, liver, placenta, kidneys and lungs.
- Imprint Gene
- Fetal Tissue
- Antisense Transcript
- Paternal Allele
- Allele 473G
Imprinted genes are specific autosomal genes that show a non-random monoallelic expression depending upon their parent of origin [1, 2]. It is estimated that the human genome contains 100–300 imprinted genes [3, 4]. A catalogue of the imprinted genes maintained at the University of Otago, New Zealand  lists approximately 44 human genes. In addition, the catalogue lists six more genes whose imprinting status is disputable.
PCR-SSCP analysis performed on genomic DNA samples from 17 abortuses showed heterozygosity in abortus no. 2 and no. 9 only with the primer set 22F/22R (Fig. 1B). The other two primer sets did not show heterozygosity in 17 abortuses (data not shown). DNA sequence analysis revealed that the heterozygosity in these abortuses was due to a G>A substitution at nucleotide position 473 (c.473G>A) (Fig. 1C). We have designated the two alleles as 473G and 473A, the wild type being 473G (Fig. 1C).
In order to determine if both or only one allele of this gene is expressed, PCR-SSCP analysis was performed using cDNA samples from different tissues of abortus nos. 2 and 9. The analysis showed expression of the allele 473G only in five tissues from abortus no. 2 (Fig. 1D) indicating that this gene was imprinted. Since the mother was homozygous for the expressed allele 473G and the abortus was heterozygous, the imprinted allele in this abortus must be derived from its father. This suggested imprinting of the paternal allele of the SLC22A1LS gene. The monoallelic expression of this gene was further confirmed in brain, lungs, liver and kidneys from another heterozygous abortus, no. 9 (Fig. 1E).
Based on their opposite orientation and knowing that SLC22A1L is paternally imprinted (maternally expressed), it was hypothesized that the SLC22A1LS gene, which overlaps with SLC22A1L, should be maternally imprinted . However, our results suggested that on the contrary, SLC22A1LS is paternally imprinted, just as is the case with its sense partner SLC22A1L. This pattern is similar to that observed in case of IGF2 and its antisense transcript IGF2-AS, both of which express the same (paternal) allele . These two examples suggest that expression of sense and antisense transcripts of a gene pair may be under the same imprinting or genetic control. It is also possible that the transcript of one gene could be regulating the transcription of its partner. The 'sense partner' of the SLC22A1LS gene, SLC22A1L, has been found to be mutated in a breast and rhabdomyosarcoma cell lines . Since patients with Beckwith-Wiedemann syndrome are known to be prone to a variety of tumors, it is possible that these genes have a role in the pathogenesis of this syndrome and in the etiology of other tumors including Wilm's tumor, although this possibility remains to be investigated.
We report for the first time the imprinting status of the SLC22A1LS gene located in human chromosome segment 11p15.5. The results suggest imprinting of the paternal allele of this gene in different fetal tissues.
A total of 17 spontaneous abortuses were ascertained over a period of three years in the Department of Gynecology and Obstretics, Kempegowda Institute of Medical Sciences, Bangalore. Following abortion, different fetal tissues were quickly dissected out and immediately frozen in liquid nitrogen. For permanent storage, tissue samples were stored at -80°C until further use. Peripheral blood samples from mothers of the abortuses were also collected in EDTA-Vacutainer® blood collection tubes (Beckton-Dickinson, USA). The study was approved by the tenets of the Declaration of Helsinki. The informed consent was obtained from human subjects included in this study.
Identification of heterozygosity in abortuses
PCR primers used in PCR-SSCP analysis to identify heterozygosity in genomic DNA samples from abortuses.
Allele specific expression
In order to test for the imprinted status of this gene, total RNA samples were isolated from different tissues of abortuses showing heterozygosity in SSCP analysis using a RNeasy® Protect mini kit (Qaigen Inc., USA). First-strand cDNAs were synthesized using a RevertAid™H First-Strand cDNA Synthesis Kit (MBI Fermentas Inc., Canada). In order to test for allele specific expression, cDNA samples were subjected to SSCP analysis as described above.
The work was supported by a grant from DST, New Delhi to AK and a JRF from CSIR, New Delhi to VB. We thank M. Ali for his help in manuscript preparation. We also thank Prof. V. Nanjundiah and two anonymous reviewers for their valuable suggestions to improve the manuscript.
- Reik W, Collick A, Norris ML, Barton SC, Surani MA: Genomic imprinting determines methylation of parental alleles in transgenic mice. Nature. 1987, 328: 248-251. 10.1038/328248a0.View ArticlePubMedGoogle Scholar
- Kumar A: Genomic imprinting: mom and dad (epi)genetics. J Biosci. 1999, 24 (1): 4-6.View ArticleGoogle Scholar
- Barlow DP: Gametic imprinting in mammals. Science. 1995, 270: 1610-1613.View ArticlePubMedGoogle Scholar
- Ke X, Thomas NS, Robinson OD, Collins A: A novel approach for identifying imprinted genes through sequence analysis of imprinted and control genes. Hum Genet. 2002, 111: 511-520. 10.1007/s00439-002-0822-3.View ArticlePubMedGoogle Scholar
- Catalogue of the imprinted genes at University of Otago, New Zealand. [http://cancer.otago.ac.nz/IGC/Web/home.html]
- Ledbetter DH, Engel E: Uniparental disomy in humans: development of an imprinting map and its implication for prenatal diagnosis. Hum Mol Genet. 1995, 4: 1757-1764.PubMedGoogle Scholar
- Gardner RJ, Mackay DJG, Mungall AJ, Polychronakos C, Siebert R, Shield JPH, Temple KI, David O, Robinson DO: An imprinted locus associated with transient neonatal diabetes mellitus. Hum Mol Genet. 2000, 9: 589-596. 10.1093/hmg/9.4.589.View ArticlePubMedGoogle Scholar
- Reid LH, Davies C, Cooper PR, Crider-Miller SJ, Sait SN, Nowak NJ, Evans G, Stanbridge EJ, deJong P, Shows TB, Weissman BE, Higgins MJ: A 1-Mb physical map and PAC contig of the imprinted domain in 11p15.5 that contains TAPA1 and the BWSCR1/WT2 region. Genomics. 1997, 43 (3): 366-375. 10.1006/geno.1997.4826.View ArticlePubMedGoogle Scholar
- Cooper PR, Smilinich NJ, Day CD, Nowak NJ, Reid LH, Pearsall RS, Reece M, Prawitt D, Landers J, Housman DE, Winterpacht A, Zabel B-U, Pelletier J, Weissman BE, Shows TB, Higgins MJ: Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain. Genomics. 1998, 49: 38-51. 10.1006/geno.1998.5221.View ArticlePubMedGoogle Scholar
- Schwienbacher C, Sabbioni S, Campi M, Veronese A, Bernardi G, Menegatti A, Hatada I, Mukai T, Ohashi H, Barbanti-Brodano G, Croce CM, Negrini M: Transcriptional map of 170-kb region at chromosome 11p15.5: identification and mutational analysis of the BWR1A gene reveals the presence of mutations in tumor samples. Proc Natl Acad Sci USA. 1998, 95: 3873-3878. 10.1073/pnas.95.7.3873.PubMed CentralView ArticlePubMedGoogle Scholar
- Okutsu T, Kuroiwa Y, Kagitani F, Kai M, Aisaka K, Tsutsumi O, Kaneko Y, Yokomori K, Surani MA, Kohda T, Kaneko-Ishino T, Ishino F: Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus in Wilms' tumors. J Biochem. 2000, 127: 475-483.View ArticlePubMedGoogle Scholar
- Kumar A, Wolpert C, Kandt RS, Segal J, Pufky J, Roses AD, Pericak-Vance MA, Gilbert JR: A de novo frame-shift mutation in the tuberin gene. Hum Mol Genet. 1995, 4 (8): 1471-1472.View ArticlePubMedGoogle Scholar
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