brief communications nature genetics • volume 30 • april 2002 365 Sotos syndrome (OMIM *117550) is a neurological disorder characterized by overgrowth from the prenatal stage through childhood, with advanced bone age, an unusual face with large skull, acromegalic features and pointed chin, occasional brain anomalies and seizures, and mental retardation. We encountered an individual with Sotos syndrome harboring the translocation 46,XX,t(5;8)(q35;q24.1) 1 . By constructing a BAC/PAC/cosmid contig covering the breakpoint, we found a partial genomic sequence homologous to mouse Nsd1 (ref. 2) located near the breakpoint (Fig. 1a). Isolation and characterization of human NSD1 revealed that it has an open reading frame of 8,088 bp, consists of 23 exons and is expressed in the human fetal brain, skeletal muscle, kidney and other tissues 3 . NSD1 is translated as a protein of 2,696 amino acids. The 5q35 breakpoint of the affected individual was located within NSD1, as seen by fluores- cence in situ hybridization (FISH) analy- sis using c6B (a cosmid subclone made from RP1-118m12) that showed split sig- nals on both der(5) and der(8) chromo- somes with almost equal signal intensity (data not shown). We identified four different de novo point mutations of NSD1 in 4 of 38 indi- viduals with Sotos syndrome for whom genomic DNA was available (Fig. 1b). They included a nonsense mutation (1310C→G) in exon 5, a one-base dele- tion (3536delA) in exon 5, a one-base insertion (5998insT) in exon 19, and a base substitution (6151+1G→A) at the splice donor site in intron 20. The 1310C→G mutation is predicted to lead to a truncation of NSD1 (Ser437X), the 3536delA leads to a truncation mutation (1,217 aa) and the 5998insT mutation results in a truncation mutation (2,007 aa). The splice site mutation (6151+1G→A) was confirmed to skip exon 20, resulting in a truncated protein with only nine amino acids added after exon 19 (2,012 aa; data not shown). In addition, FISH analysis revealed a com- mon 2.2-Mb deletion in 19 individuals and a smaller deletion in one individual from a total of 30 affected individuals whose metaphase or interphase cells were available (Fig. 1c,d). These deletions involve the whole NSD1. Together with the data for point mutations, these data indicate that 77% of the individuals with Sotos syndrome in our study have either deletions or point mutations. All of the Haploinsufficiency of NSD1 causes Sotos syndrome Published online: 18 March 2002, DOI: 10.1038/ng863 We isolated NSD1 from the 5q35 breakpoint in an individual with Sotos syndrome har- boring a chromosomal translocation. We identified 1 nonsense, 3 frameshift and 20 submicroscopic deletion mutations of NSD1 among 42 individuals with sporadic cases of Sotos syndrome. The results indicate that haploinsufficiency of NSD1 is the major cause of Sotos syndrome. c6B CTC-286c20 (AC027314 ) FGFR 4 JA Z NSD1 CTC-549a4 (AC008570 ) RP3-378o2 3 R P1-32c 5 R P3-469e8 RP1-251c21 RP1-118m1 2 CTC-2301a4 c6A c2B c4D der(5) normal 5 PAC/BAC cosmid genome s equence gene cen tel BREAKPOINT 1, 2 3 chromos ome 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 18 19 20 21 22 23 NID -L NID + L PWWP I P HD-I P HD-I I PWWP I I SE T P HD-II I AAGGGGTCAAAGAACC TGA 1310C→G, Ser437X CAGATTTAAAGAGAAAGAA CAGATTTAAAGGAAAGAAA A 3536delA AAAGCAGGTAAGAAT A 6151+1G→A 5998insT TATATGCTCACCCTAGAC A TATATGCTC T ACCCTAGAC ce n 19 5 19 6 197 198 Mb tel known genes FISH probes HSPC111 KIAA1191 FLJ11561 B4GALT7 FLJ10404 ABS FLJ22570 ENIGMA DBN1 MGC10772 GPRK6 F12 SLC34A1 JAZ FGFR4 NSD1 LOC51720 SNCB HK3 FLJ20124 KIAA1100 RP11-103O5 RP11-147K7 RP1-118M12 RP11-606E24 RP11-1006E8 RP11-1107B24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fig. 1 NSD1 mutations in individuals with Sotos syn- drome. a, BAC/PAC/cosmid map spanning the 5q35 breakpoint. Red and blue horizontal lines indicate clones spanning the breakpoint (detected by FISH analysis) and complete genomic sequences, respec- tively. Arrows indicate genes, and green boxes below NSD1 represent exons 1, 2 and 3. b, Genomic structure of NSD1 and four point mutations found in individuals with Sotos syndrome. Open and gray boxes and arrowheads indicate exons, the 5’ and 3’ untranslated regions, and start and stop codons, respectively. Specific domains are indicated by col- ored boxes, and sequence traces disclose mutations in lower row. c, FISH analysis of the affected individ- ual harboring the deletion. Absence of a FISH signal for RP1-118m12 containing NSD1 (green) along with the presence of 5pter signals (red) on the individual’s chromosome 5 is apparent. d, Summary of FISH dele- tions in 20 affected individuals. Known genes, probes used and their genomic locations are indicated in the upper row. Numbers (1−20) and black and blue lines represent affected individuals, regions without dele- tion and those regions deleted, respectively. a b c d © 2002 Nature Publishing Group http://genetics.nature.com