European Journal of Human Genetics (2006) 14, 838–845 & 2006 Nature Publishing Group All rights reserved 1018-4813/06 $30.00 www.nature.com/ejhg ARTICLE Differences in methylation patterns in the methylation boundary region of IDS gene in hunter syndrome patients: implications for CpG hot spot mutations Shunji Tomatsu*,1, Kazuko Sukegawa2, Georgeta G Trandafirescu1, Monica A Gutierrez1, Tatsuo Nishioka1, Seiji Yamaguchi3, Tadao Orii2, Roseline Froissart4, Irene Maire4, Amparo Chabas5, Alan Cooper6, Paola Di Natale7, Andreas Gal8, Akihiko Noguchi1 and William S Sly9 1Department of Pediatrics, Saint Louis University, Pediatric Research Institute, St Louis, MO, USA; 2Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan; 3Department of Pediatrics, Shimane University, Shimane, Japan; 4Centre d’Etude des Maladies Me´taboliques, Hoˆpital Debrousse, Lyon France; 5Institut de Bioquimica Clinica, Barcelona, Spain; 6Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Great Britain, UK; 7Department of Biochemistry and Medical Biotechnologies, University of Naples, Federico II, Italy; 8Institut fur Humangenetik, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Germany; 9Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome. European Journal of Human Genetics (2006) 14, 838–845. doi:10.1038/sj.ejhg.5201615; published online 12 April 2006 Keywords: mucopolysaccharidosis II; methylation; CpG dinucleotide; IDS; recurrent mutation *Correspondence: S Tomatsu, Department of Pediatrics, Saint Louis Contract grant sponsor: Austrian Research Society for Mucopolysacchar- University Pediatric Research Institute, 3662 Park Ave., St Louis, idoses and Related Diseases, German MPS Society, Italian MPS Society, MO 63110-2586, USA. and International Morquio Organization (Carol Ann Foundation). Tel: þ 1 314 577 5623, ext. 6213; Fax: þ 1 314 577 5398; Received 14 September 2005; revised 14 February 2006; accepted 15 E-mail: [email protected] February 2006; published online 12 April 2006 Methylation studies in MPS II S Tomatsu et al 839 Introduction and characterized.14 – 16 At least 307 independent point Methylation of cytosine is the only known endogenous mutations (160 different nucleotide changes) have been modification of DNA in mammals and occurs by the described in Hunter patients17 – 28,32 (mutations are cited in enzymatic addition of a methyl group to the carbon-5 www.hgmd.org).28 position of cytosine. In 98% of the genome, CpGs are At least 40% of point mutations were C-to-T transitional present approximately once per 80 dinucleotides. In mutations at CpG sites.28 The 56 CpGs represent only 3.6% contrast, CpG islands, which comprise 1–2% of the of the IDS gene sequence. These finding indicate that the genome, are approximately 200 bp to several kb in length incidence of transitional mutational events at CpG sites in and have a frequency of CpGs approximately five times the IDS gene is about 30 times greater than expected. The greater than the genome as a whole.1,2 methylation pattern in five normal healthy males showed The epigenetic modification of eukaryotic DNA by the that cytosine methylation at the CpG sites was extensive, methylation of cytosine residues modulates access to and except for sites in the promoter region to exon 3.29 The regulation of genetic information.3 This process is impli- CpG sites in exons 4–9, where over 90 independent cated in the regulation of gene expression, oncogenesis, transitional mutations were found, were completely imprinting, and X-inactivation. Moreover, one of the most methylated in healthy male control samples. Only two significant and well-known aspects related to DNA methy- transitional events were observed in exons 1 and 2, which lation is mutability. DNA methylation is a major con- were unmethylated or hypomethylated. These findings tributor to point mutations leading to human genetic support the importance of CpG cytosine methylation in disease as a consequence of deamination of 5-methylcyto- predisposition to C-to-T transition mutation in the IDS sine present within CpG dinucleotides.4,5 In spite of the gene. Exceptions were the CpG sites in exon 3, which were relative paucity of the number of CpG dinucleotides and unmethylated or hypomethylated in healthy male con- the presence of a sophisticated repair system, more than trols, yet associated with a high rate of transitional one-third of all point mutations causing human genetic mutations in Hunter patients. This lack of correlation diseases are derived from C-to-T or G-to-A transitions at a between transitional mutations at CpG sites in exon 3 and CpG site.4 the methylation status in this region in normal healthy The methylation status of specific CpG sites does exhibit males might be explained if there was some polymorphism significant inter-individual variation, although differences in the degree of methylation of CpG sites in exon 3 in in site-specific methylation patterns between different humans and CpG mutations in exon 3 occur predomi- ethnic groups have not so far been apparent in studies of nantly in those patients who show greater than normal a variety of DNA sequences.6–8 Most methylation analyses methylation in this region. in housekeeping genes, imprinted genes, or oncogenes To test this hypothesis, we compared methylation have been limited to the 50 flanking sequencers. However, patterns of specific CpG dinucleotides between normal direct and specific observations of CpG methylation at the healthy males and Hunter patients with CpG transitional sites of recurrent mutations in a portion of genes have been mutations. Our results support the hypothesis that the described.9–11 To gain a better understanding of the CpG sites in this region are more highly methylated in frequent transitional mutation events at CpG dinucleo- exon 3 of Hunter patients with mutations in this region. tides, studies on pattern of cytosine methylation covering the promoter region and the whole coding region have been performed.12,13 These studies showed a high muta- Materials and methods tion frequency at methylated CpG sites and definition of Sample preparation the boundaries between unmethylated and methylated Genomic DNAs of 11 Hunter patients with the transitional regions. However, to date no study has compared differ- mutations and five normal healthy males were isolated ences at the methylation level of specific CpG sites from peripheral blood samples and analyzed to determine between the normal population and a patient group in the distribution and the percentage of methylated cytosine which CpG transitional mutations lead to an inherited residues at the IDS gene locus encompassing the upstream genetic disease. region of initial codon ATG and exon 1 to a part of intron Hunter syndrome (or mucopolysaccharidosis type II) is 3. The 11 patients included one with p.R8X in exon 1 an X-linked recessive disease caused by the deficiency of (Patient 11, 28 years old, referred as JS27), four with p.A85T the enzyme, iduronate-2-sulphatase (IDS; EC3.1.6.13, (Patient 4, 15 years old, referred as HT65;25 Patient 9, 6 MIM#309900). In humans, this deficiency leads to accu- years old, cousin of HT65; Patient 10, 5 years old, cousin of mulation of heparan sulfate and dermatan sulfate in HT65; Patient 23, 9 years old, referred as H624), three with lysosomes and their excretion in the urine. Clinically, p.P86L (Patient 1, fetus, referred as Fe 1;30 Patient 14, 1 year the phenotype varies from severe to mild, depending old, referred as CC;27 Patient 28, 1 year old, referred as no. on the different mutations at the IDS gene. IDS cDNA and 623), and three (Patient 2, fetus, referred as Fe 2;30 Patient its full genomic DNA sequences have been isolated 17, 6 years old, referred as No. 7;22 Patient 33, 2 years old, European Journal of Human Genetics Methylation studies in MPS II S Tomatsu et al 840 23 F6 referred as no. 8 ) with p.R88H in exon 3. Patients 4, 9, F5 F4 and 10 were in the same family tree. Mutation analysis of F3 22 – 25,27,30 the patients was described previously. Peripheral F1 F2 genomic DNAs from five unrelated healthy males were also isolated. 1 2 3 aF1 aF2 aF3 Sodium bisulfite treatment and sequencing aF4 aF5 Bisulfite conversion of genomic DNA was performed using aF6 the protocol described previously with minor modifica- tion,31 as we recently described for analysis of the b- 12,29 glucuronidase and IDS genes.