Exploring the unique function of imprinting control centers in the PWS/AS-responsible region: finding from array-based methylation analysis in cases with variously sized microdeletions

Background Human 15q11–13 is responsible for Prader-Willi syndrome (PWS) and Angelman syndrome (AS) and includes several imprinted genes together with bipartite elements named AS-IC (imprinting center) and PWS-IC. These concertedly confer allele specificity on 15q11–13. Here, we report DNA methylation status of 15q11–13 and other autosomal imprinted differentially methylated regions (iDMRs) in cases with various deletions within the PWS/AS-responsible region. Methods We performed array-based methylation analysis and examined the methylation status of CpG sites in 15q11–13 and in 71 iDMRs in six cases with various microdeletions, eight cases with conventional deletions within 15q11–13, and healthy controls. Results We detected 89 CpGs in 15q11–13 showing significant methylation changes in our cases. Of them, 14 CpGs in the SNORD116s cluster presented slight hypomethylation in the PWS cases and hypermethylation in the AS cases. No iDMRs at regions other than 15q11–13 showed abnormal methylation. Conclusions We identified CpG sites and regions in which methylation status is regulated by AS-IC and PWS-IC. This result indicated that each IC had unique functions and coordinately regulated the DNA methylation of respective alleles. In addition, only aberrant methylation at iDMRs in 15q11–13 leads to the development of the phenotypes in our cases. Electronic supplementary material The online version of this article (10.1186/s13148-019-0633-1) contains supplementary material, which is available to authorized users.


Introduction
The chromosomal region 15q11-13 includes several genes showing monoallelic expression in a parent-of-origin-specific manner [1]. This region is responsible for Prader-Willi syndrome (PWS, OMIM #176270) and Angelman syndrome (AS, OMIM #105830). PWS is characterized by hypotonia during infancy, developmental delay, hyperphagia followed by morbid obesity, and cognitive impairment [2], and AS is associated with severe developmental delay, ataxia, and recurrent seizures [3]. Approximately 70% of PWS and AS patients have 5-7 Mb deletions affecting the 15q11-13 imprinted region ( Fig. 1). Recently, it has been reported that deletion involving the SNORD116 gene, which is one of the C/D box small nucleolar RNAs (snoRNAs), causes the phenotypes of PWS [4][5][6][7][8]. AS phenotypes arise from the loss of expression or function of the UBE3A gene, which is expressed on the maternally derived allele in mature neurons [3].
The imprinting control region (ICR) conferring parent-of-origin identity of the genes on 15q11-13 was defined according to the smallest region of overlap (SRO) found in PWS or AS individuals with rare atypical microdeletions [9]. The ICR on 15q11-13 consists of bipartite DNA elements named AS-IC (imprinting center) and PWS-IC [10]. The PWS-IC is a 4.1-kb region, which spans the SNURF/SNRPN promoter and exon 1, and includes an imprinted differentially methylated region (iDMR), which is maternally methylated and paternally unmethylated. The AS-IC is an 880-bp sequence located 35 kb centromeric of the PWS-IC and does not include iDMR. It is known that PWS-IC and AS-IC cooperate in regulating epigenetic status and allele-specific gene expression at this locus [1].
Recent advancement in methylation analysis allows genome-wide methylation studies, and it detected novel iDMRs located in regions near the SNURF/SNRPN gene and several CpG sites in the SNORD116s cluster showing a slight tendency for preferential paternal methylation [11]. The regulatory mechanism in DNA methylation of those imprinted regions by ICR has not yet been elucidated precisely.
In this study, we performed an array-based DNA methylation analysis in the cases with various deletions involving the PWS/AS region and examined the    iDMRs at regions other than the PWS/AS region to clarify the regulatory mechanism for DNA methylation in the 15q11-13 imprinted region.

Results and discussion
Six cases with atypical microdeletions in the 15q11-13 region (cases 1-6) and four with PWS and four with AS due to conventional large deletions (LD) in 15q11-13 (PWS LD and AS LD , respectively) were enrolled in this study (Table 1 and Additional file 1: Supplementary document).

Deletion size and breakpoint
Custom-built array CGH revealed a copy number loss in each case ( Fig. 2a and Additional file 2: Figure S1). This array contained approximately 38,000 probes for 15q11-q13 encompassing the imprinted region and~10,000 reference probes for other chromosomal regions (4×180K format, Design ID 032112) (Agilent Technologies, Palo Alto, CA, USA). The detailed information of probes contained in this custom-built array is shown in Additional file 3: Table S1. Cases 1 and 2 had a deletion involving both AS-IC and PWS-IC, case 3 had a deletion involving only the SNORD116s cluster, and cases 4-6 had a deletion involving only AS-IC.

Difference in the DNA methylation status between each case with various microdeletions and controls
We focused on the DNA methylation status at CpG sites on autosomal chromosomes in two groups (groups 1 and 2) and extracted methylation data at 1335 probes located in CpG sites at the 15q11-13 PWS/AS region (group 1, Additional file 4: Table S2) and at 863 probes in the iDMRs at regions other than the PWS/AS region (group 2, Additional file 5: Table S3).
CpG sites in 15q11-13 (probes in group 1) We identified 89 probes in group 1 showing significant differences in DNA methylation in at least 1 case against normal controls ( Fig. 2b and Additional file 6: Table S4).
Most probes located in the miR4508-PWS-IC region c b a Fig. 2 Genomic organization of the PWS/AS locus and alteration in DNA methylation of CpG sites in 15q11-13. a Summary of the loci and the approximate sizes of the deleted regions in cases 1-6. b Unsupervised hierarchical clustering and heat map of the 450k methylation data of 89 probes with |Δβ| > 0.2 and FDR p values < 0.01 between at least 1 case and controls are shown. The cases with AS phenotype are shown in red boxes, those with PWS phenotype in blue boxes, and the carriers and normal controls in green boxes. Blue and yellow colors indicate 0 and 1 methylation, respectively. The y-axis represents the names of iDMRs which had been known to be differentially methylated previously. Not all names of the iDMRs are shown. c The differences in β values (Δβ) of probes located in the SNORD116s and 115s clusters between each case and controls are shown using IGV (Integrated Genome Viewer, http://software.broadinstitute.org/software/igv/). The probes showing "hypermethylated" in cases are represented by yellow vertical bars, and those showing "hypomethylated" by blue ones. ICR, imprinting control region; IC, imprinting center; kb, kilobase showed hypermethylation in case 1 and PWS LD and hypomethylation in cases 4 and 5 and AS LD , respectively. Case 3 showed normal methylation status in these sites. Hierarchical clustering of the methylation status of these 89 CpGs showed that all cases other than case 3 with the SNORD116s deletion were classified into subgroups according to their phenotypes (Fig. 2b). Case 3 was clustered closer to the normal controls.
In case 2, who was the father of case 1, probes in his deleted region in PWS-IC showed hypomethylation; however, the remaining probes showed normal methylation status. In case 6, who was the mother of case 5, no CpG with aberrant methylation was found.
In addition, 14 probes in the SNORD116s cluster presented slight hypomethylation in the cases with PWS phenotype (cases 1and 3 and PWS LD ) and hypermethylation in the cases with AS phenotype (cases 4 and 5 and AS LD ) (Fig. 2c).
iDMRs at regions other than the PWS/AS region (probes in group 2) Additional file 7: Table S5 shows the results of the methylation analyses for 71 iDMRs including 828 probes in group 2 between each of cases 1-6 and normal controls. We detected no iDMR with an abnormal methylation pattern.
In this study, we examined the methylation status of CpG sites in 15q11-13 and in 71 iDMRs at regions other than the PWS/AS region using methylation array for the cases with various deletions involving the PWS/ AS region. This study provides several notable findings. First, we clarified CpG sites and regions in which the methylation status was regulated by AS-IC and PWS-IC in 15q11-13. Most abnormally methylated CpG sites in cases with deletions were located within or very close to the regions reported in previous studies [11][12][13]. Based on the results from our patients and a previously reported PWS patient with a microdeletion involving only PWS-IC, we present a hypothetical model for the regulation of DNA methylation at the 15q11-13 imprinted region in Fig. 3. We suggest regulatory mechanisms of this region as follows: (1) maternal pattern (methylated) at CpGs in the upstream region of the ICR was the default state (see normal state and case 2), (2) AS-IC was required for the methylation of PWS-IC on the maternally derived allele (compare case 6 with cases 4 and 5), (3) the unmethylated PWS-IC led to the unmethylated status of CpGs in the upstream of the ICR even on the maternally derived allele (see cases 4 and 5), and (4) the methylation patterns of several CpGs in the SNORD116s clusters were also regulated by both AS-IC and PWS-IC. Several CpGs in the SNORD116s cluster were preferentially methylated on the paternally inherited allele (Fig. 2c) [11]. The methylation pattern in the SNORD116s cluster was different between case 1 and the PWS case lacking only PWS-IC [14]. These results indicate that both AS-IC and unmethylated PWS-IC on the paternally derived allele function independently as regulators of the methylation status of CpGs in the SNORD116s cluster. Furthermore, several CpGs in the SNORD116s cluster were methylated in cases 4 and 5 on the maternally derived allele. This result indicates that unmethylated PWS-IC was needed for the methylation of the CpGs in the SNORD116s cluster independently on the parental origin. In the cases with large deletions involving the entire region of 15q11-13 (Fig. 3c), abnormal methylation patterns were simply due to the loss of the paternally or maternally derived allele in PWS or AS patients, respectively.
Second, this study demonstrated that PWS/AS case with various deletions had normal methylation status in known iDMRs at regions other than 15q11-13. This result indicates that the development of phenotypes in our cases was not caused by aberrant methylation changes at iDMRs at regions other than 15q11-13. In addition, our study and previous studies using other methylation analyses showed a normal methylation pattern at PWS-IC in patients with deletions only including the SNORD116s cluster [4][5][6][7]. It remains to be clarified how the deletion of SNORD116s contributes to the development of PWS phenotypes. Previous studies reported several findings regarding long non-coding RNAs (sno-lncRNAs) including SNORD116s: (1) some functional units, 116 host genes (116HG) and snoRNA-related sno-lncRNAs, were processed from the SNORD116s cluster in human tissues or cells [15,16], (2) Fox-family splicing regulators are bound to sno-lncRNAs and altered splicing patterns of genes are related to neuronal development in human ES cells [17], and (3) there was a Snord116-dependent diurnal rhythmic DNA methylation in the mouse cortex [18]. Thus, the loss of the expression of SNORD116s may lead to PWS-relevant phenotypes, such as abnormalities of energy metabolism and diurnal rhythm [17,18].
It remains to be elucidated how AS-IC and PWS-IC establish the allele-specific methylation patterns in neighboring CpGs. Recently, it was reported that transcripts from an oocyte-specific promoter in AS-IC are needed for the acquisition of maternal DNA methylation patterns in PWS-IC in human oocytes [19]. However, there has apparently been no study examining the direct association between AS-IC and the SNORD116s cluster, although unmethylated PWS-IC on the paternal allele physically interacts with paternally expressed genes in the upstream region of ICR [14]. In addition, how unmethylated PWS-IC establishes paternal methylation patterns at CpGs in the PWS/AS region in cis and how PWS-IC interacts with CpG sites at the SNORD116s cluster on paternally derived alleles remain unknown.
In summary, we performed an array-based methylation study in cases with various-sized microdeletions in the PWS/AS region on maternally or paternally derived chromosome 15. We identified CpG sites and regions in which the DNA methylation status is regulated by ICR. Our results enabled us to speculate on the regulatory mechanism for the establishment of the methylation status of local CpG sites or regions in 15q11-13. Moreover, we demonstrated that the development of phenotypes in the PWS/AS patients with deletions encompassing ICR and/or the SNORD116s cluster was not mediated by aberrant methylation changes at iDMRs at regions other than the PWS/AS regions. Further investigation will be In the second left case with a deletion involving only PWS-IC in the paternally inherited allele, the methylation pattern in the SNORD116s cluster is depicted according to the results of a previous report [14]. c DNA methylation pattern in cases with large deletions involving the entire PWS/AS region. AS-IC and PWS-IC seemed to control DNA methylation status cooperatively or separately at the PWS/AS region necessary to fully elucidate how paternal deletion of SNORD116s causes PWS phenotypes.

Subjects
Six cases with atypical microdeletions in the 15q11-13 region (cases 1-6) and four with PWS LD and four with AS LD due to conventional large deletions were enrolled in this study (Figs. 1 and 2a, Table 1, Additional file 1: Supplementary document, and Additional file 8: Figure S2). Genomic DNAs from leukocytes in these cases and healthy controls were utilized for this study. Methyl ation-specific multiplex ligation-dependent probe amplification (MS-MLPA; ME030, MRC-Holland, Amsterdam, Netherlands) was performed in all cases to examine the deleted region and DNA methylation status in 15q11-13 (data not shown). All cases showed normal karyotype. Clinical manifestations of cases are shown in the supplementary document. Healthy children (n = 11) and adults (n = 24) were involved as normal controls.

Copy number analysis
We designed a custom-built array-based comparative genomic hybridization (aCGH) and utilized this aCGH to narrow down the deleted regions in cases 1-6 (4×180K format, Design ID 032112, Agilent Technologies, Palo Alto, CA, USA). The detailed information of probes contained in this array is shown in Additional file 3: Table S1. The procedure was as described in the manufacturer's instructions.