No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.
BACKGROUND
Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease.


RESULTS
DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing.


CONCLUSIONS
As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.
Bernier-Latmani
Jeremiah
J
Experimental Pathology Division, Institute of Pathology, University of Lausanne, Lausanne, Switzerland.
Baumer
Alessandra
A
Shaw
Phillip
P
D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons; D018392:Genomic Imprinting; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D056730:Silver-Russell Syndrome / Q000235:genetics*
C456540:CTCFL protein, human; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated
Nat Struct Mol Biol. 2009 Mar;16(3):304-311
19234465
J Med Genet. 2008 Apr;45(4):193-9
18156438
Development. 2002 Apr;129(8):1983-93
11934864
Biotechniques. 2006 Aug;41(2):177-81
16925019
Nature. 2004 Jun 24;429(6994):900-3
15215868
PLoS Biol. 2006 Oct;4(11):e355
17048991
J Comput Biol. 1997 Fall;4(3):311-23
9278062
Cell. 1991 Feb 22;64(4):849-59
1997210
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6806-11
12011441
Nature. 2000 May 25;405(6785):486-9
10839547
Mol Cell. 2008 Oct 10;32(1):1-9
18851828
J Clin Endocrinol Metab. 2009 Feb;94(2):579-87
19017756
J Biol Chem. 2004 Jun 25;279(26):27816-23
15105426
Trends Genet. 2008 Apr;24(4):195-204
18329128
J Med Genet. 2003 Apr;40(4):e44
12676917
Nat Genet. 2003 Jan;33(1):66-9
12461525
Nat Genet. 2005 Sep;37(9):1003-7
16086014
Nature. 1991 May 9;351(6322):153-5
1709450
Nucleic Acids Res. 2007;35(21):7372-88
17962299
Clin Genet. 2003 Sep;64(3):252-4
12919141
J Med Genet. 2009 Mar;46(3):192-7
19066168
Mol Cell Biol. 1987 Aug;7(8):2745-52
3670292
Nucleic Acids Res. 2003 Jul 1;31(13):3568-71
12824367
Eur J Med Genet. 2008 Jul-Aug;51(4):322-4
18585117
Am J Med Genet. 2001 Apr 1;99(4):335-7
11252004
Am J Med Genet. 2001 Dec 1;104(3):225-31
11754049
J Med Genet. 2003 May;40(5):e65
12746419
Genet Test. 2003 Fall;7(3):259-63
14642004