Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.

Sotos syndrome (SoS) is a congenital dysmorphic disorder characterized by overgrowth in childhood, distinctive craniofacial features, and mental retardation. Haploinsufficiency of the NSD1 gene owing to either intragenic mutations or microdeletions is known to be the major cause of SoS. The common approximately 2.2-Mb microdeletion encompasses the whole NSD1 gene and neighboring genes and is flanked by low-copy repeats (LCRs). Here, we report the identification of a 3.0-kb major recombination hotspot within these LCRs, in which we mapped deletion breakpoints in 78.7% (37/47) of patients with SoS who carry the common microdeletion. The deletion size was subsequently refined to 1.9 Mb. Sequencing of breakpoint fragments from all 37 patients revealed junctions between a segment of the proximal LCR (PLCR-B) and the corresponding region of the distal LCR (DLCR-2B). PLCR-B and DLCR-2B are the only directly oriented regions, whereas the remaining regions of the PLCR and DLCR are in inverted orientation. The PLCR, with a size of 394.0 kb, and the DLCR, with a size of of 429.8 kb, showed high overall homology (approximately 98.5%), with an increased sequence similarity (approximately 99.4%) within the 3.0-kb breakpoint cluster. Several recombination-associated motifs were identified in the hotspot and/or its vicinity. Interestingly, a 10-fold average increase of a translin motif, as compared with the normal distribution within the LCRs, was recognized. Furthermore, a heterozygous inversion of the interval between the LCRs was detected in all fathers of the children carrying a deletion in the paternally derived chromosome. The functional significance of these findings remains to be elucidated. Segmental duplications of the primate genome play a major role in chromosomal evolution. Evolutionary study showed that the duplication of the SoS LCRs occurred 23.3-47.6 million years ago, before the divergence of Old World monkeys.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.