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Identification of eight novel NSD1 mutations in Sotos syndrome
  1. J Kamimura1,2,3,
  2. Y Endo1,
  3. N Kurotaki1,
  4. A Kinoshita1,3,
  5. N Miyake1,3,
  6. O Shimokawa4,
  7. N Harada1,3,4,
  8. R Visser1,6,7,
  9. H Ohashi8,
  10. K Miyakawa9,
  11. J Gerritsen10,
  12. A M Innes10,
  13. L Lagace11,
  14. M Frydman12,
  15. N Okamoto13,
  16. R Puttinger14,
  17. S Raskin15,
  18. B Resic16,
  19. V Culic16,
  20. K Yoshiura1,3,
  21. T Ohta3,5,
  22. T Kishino3,5,
  23. M Ishikawa2,
  24. N Niikawa1,3,
  25. N Matsumoto1,3
  1. 1Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
  2. 2Department of Obstetrics and Gynecology, Asahikawa Medical College, Asahikawa, Japan
  3. 3CREST, Japan Science and Technology Corporation, Kawaguchi, Japan
  4. 4Kyushu Medical Science Nagasaki Laboratory, Nagasaki, Japan
  5. 5Division of Functional Genomics, Research Center for Frontier Life Sciences, Nagasaki University, Nagasaki, Japan
  6. 6International Consortium for Medical Care of Hibakusha and Radiation Life Science, The 21st Century COE (Center of Excellence), Nagasaki, Japan
  7. 7Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands
  8. 8Division of Medical Genetics, Saitama Children’s Medical Center, Saitama, Japan
  9. 9Division of Pediatrics, Niigata City General Hospital, Niigata, Japan
  10. 10Department of Medical Genetics, University of Calgary, Calgary, Canada
  11. 11East Central Health, Alberta, Canada
  12. 12Danek Gertner Institute of Human Genetics, Chaim Sheba Medical Center, Tel-Aviv University, Ramat Gan, Israel
  13. 13Department of Planning and Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan
  14. 14Klinsche Genetik, St. Johanns-Spital, Salzburg, Austria
  15. 15Laboratorio Genetika, Curitiba Parana, Brazil
  16. 16Clinical Hospital Split, Pediatrics Clinic, Department of Developmental Neurology and Medical Genetics, Spinciceva, Croatia
  1. Correspondence to:
 Dr N Matsumoto
 Department of Human Genetics, Nagasaki University School of Medicine, Sakamoto 1-12-4, Nagasaki 852-8523, Japan;

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Sotos syndrome or cerebral gigantism (SoS, OMIM #117550) is a well-known disorder characterised by overgrowth with advanced bone age, craniofacial anomalies, developmental delay, and occasional seizures.1,2 A typical face has a large head circumference, frontal bossing with high anterior hairline, down slanting of palpebral fissures, flat nasal bridge, and prominent jaw.3 The hands and feet are usually large. Height and weight tends to normalise in adulthood.4 EEG abnormalities, hypotonia, strabismus, congenital heart defects, kyphoscoliosis, and cancer have also been noted.3,5–10 Since the original report in 1964,1 more than 300 affected cases have been reported. Most cases are sporadic, while several familial cases have been described, suggesting that SoS is an autosomal dominant disorder.8,11–17

We have previously isolated the nuclear receptor SET domain containing gene 1 (NSD1) from the 5q35 translocation breakpoint in a Japanese SoS patient with t(5;8)(q35; q24.1).18,19NSD1 encodes 2696 amino acids (GenBank accession no. AF395588), and the gene has several putative functional domains, such as NID−L, NID+L, SET, SAC, PWWP-I, PWWWP-II, PHD-I, PHD-II, and PHD-III, suggesting that the NSD1 protein may be associated with chromatin mediated transcriptional regulation.19–24 In 42 Japanese sporadic cases of SoS, we identified 20 submicroscopic deletions including the entire NSD1 gene and four point mutations, the data indicating that SoS is caused by haploinsufficiency of NSD1.25 Recently, a UK group reported 29 novel NSD1 point mutations and only three microdeletions in 37 typical SoS and 13 SoS-like patients, and suggested that NSD1 intragenic mutations instead of microdeletions were the major cause of SoS.26

In this study, we validated the spectrum of NSD1 intragenic mutations among 30 newly collected SoS patients.


The subjects studied included 13 Japanese and 17 non-Japanese patients with SoS. No patient with Weaver synrome was included in this study. The 17 non-Japanese cases comprised four Canadians including two Hutterite, three each of Brazilians, Germans, and Italians, and one each of Israeli-Arab, Israeli, Austrian, and Croatian. Three main features were considered at the clinical diagnosis: (a) typical craniofacial dysmorphology including macrocephaly, high anterior hairline, down slanting palpebral fissures, and prominent jaw, (b) developmental delay (intelligence quotient or development quotient <80), and (c) history of overgrowth (height and weight >+2 SD). Advanced bone age was not evaluated, because sufficient data were not available. Adequate clinical information was available in 22 patients. In the other eight cases, only limited information was provided for this study. All patients were referred to us after microdeletions were ruled out by fluorescent in situ hybridisation analysis using a P1 derived artificial chromosome probe (RP11-118M12), as described previously.20 Six patients (SoS123a (daughter) and SoS123b (mother) from a Japanese family, SoS153a (son) and SoS153b (father) from a Canadian-Hutterite family, and SoS152a (brother) and SoS152b (sister) from an Italian family) were originally suspected to be familial.

Key points

  • Sotos syndrome (SoS) (OMIM #117550) is an autosomal dominant overgrowth disorder with developmental delay, typical dysmorphic craniofacial features, and advanced bone age. The syndrome is caused by haploinsufficiency of NSD1.

  • In the Japanese population, a common microdeletion including NSD1 accounts for about a half of SoS patients. In contrast, only 6% of SoS or SoS-like cases in the UK were shown to have a deletion, but 58% had NSD1 point mutations. Thus far, 38 intragenic mutations of NSD1 have been reported.

  • To investigate the spectrum of NSD1 point mutations in SoS patients, we performed NSD1 mutation analysis by direct sequencing in 30 subjects whose microdeletions were already ruled out by fluorescent in situ hybridisation analysis.

  • We identified eight intragenic mutations: one insertion, thre small deletions, and four nonsense mutations. All mutations were novel and were predicted to cause protein truncations. The data obtained thus far do not support the presence of hot spots for NSD1 point mutations.

  • Low frequency of NSD1 mutations in this series may be explained in part by a significant portion of collected patients showing atypical SoS or Sotos-like syndrome, and possible genetic heterogeneity.

Peripheral blood samples were collected from the patients after obtaining informed consent, and genomic DNA was extracted according to a standard method. The study was approved by the ethics committee of Nagasaki University.

The 22 exons covering the NSD1 coding region (exons 2–23) as well as intron–exon boundaries were amplified by PCR. The primer sequences designed at our previous study are available for a request.25 PCR was cycled 35 times at 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min in volume of 25 μl, containing 1× PPCR buffer with 2 mmol/l MgCl2, 0.2 mmol/l each dNTP, 1 μmol/l each primer and 2.5 U Taq polymerase. Amplified PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA, USA), sequenced on both strands with the BigDye Terminator Cycle Sequencing Ready Reaction kit (version 3.0; PE Applied Biosystems, Foster City, CA, USA), according to the manufacturers’ protocols, and analysed on an ABI 3100 automated DNA sequencer with the sequence analysis software and the AutoAssembler software (version 2.1.1) (all PE Applied Biosystems).


We identified eight different intragenic mutations of NSD1 in the 30 SoS patients analysed (table 1). All were heterozygous mutations. Bidirectional sequence analyses were carried to be sure that all heterozygous mutations are authentic. These mutations included one insertion (4769insT), three small deletions (1807delT, 2053–2057del, and 3273delT), and four nonsense mutations (1130G→A (W377X), 3886A→T (K1296X), 3964C→T (R1322X), 5229G→A (W1743X)). All mutations were novel and observed through exons 4 to 15 (fig 1). All mutations found are predicted to result in protein truncations. One insertion (4769insT) and two nonsense mutations (Lys1296X, Arg1322X) were judged after sequencing their parental DNA to have occurred de novo. Ages of patients with and without mutations were 3 months to 14 years (mean 6.4 years) and 9 months to 40 years (mean 13.2 years), respectively.

Table 1

Summary of NSD1 mutations identified in 30 patients with Sotos syndrome

Figure 1

Schematic representation of the genomic structure of NSD1, and location of detected mutations. Open and grey/filled boxes depict exons and functional domains of NSD1, respectively. Arrows indicate mutations.


NSD1 intragenic mutations were found in eight (27%) of 30 newly collected SoS patients. Microdeletions had already been ruled out in this series of patients before the sequence analysis. The present data, combined with those from previous studies,25–28 give a total of 38 NSD1 intragenic mutations distributed in exons 2, 4–7, 10, 13–16, 18–20 and 22–23. The data do not support the presence of hot spots for NSD1 point mutations. Different types of mutations were found, insertion, deletion, and nonsense mutations, and all may lead to protein truncations.

Mutations were not found in the other 22 patients (73%). Among these, adequate clinical information was available in 15 patients. Nine patients out of the 15 showed only one (a typical craniofacial feature) or two of three main features of SoS, suggesting that 22 patients without any NSD1 mutations might have included many atypical SoS or Sotos-like patients. Instead, seven patients with mutations whose clinical information was fully available showed all three features. Erroneous diagnosis might also be possible, as the patients in this study were referred to us by a number of physicians. The average age of patients with mutations was younger than that of patients without any mutations. We assume that correct diagnosis in infancy and childhood is easier than in adulthood because overgrowth and developmental delay are prominent. Other mutational events that cannot be detected by current methods may be observed in some of our patients, such as intronic mutations that affect transcription, silent mutations including exon skipping, or NSD1 promoter mutations. Alternatively, another SoS locus might exist.

Among six patients suspected to be familial, only one patient (SoS153a) showed an NSD1 mutation, 3886A→T (K1296X). His father (SoS153b) who showed a similar craniofacial feature, but no developmantal delay or overgrowth, had no such abnormality. SoS123a (daughter) with all three features and SoS123b (mother) with a similar phenotype but no overgrowth in a Japanese family, and SoS152a (brother) and SoS152b (sister) both showing all three features in an Italian family, presented with normal NSD1 sequences. Thus, we could not identify any transmitted NSD1 mutations in the three families. To date, only one point mutation (896delC) in a father and son from a Finnish family has been reported.27 Previous studies suggested reproductive reduction in SoS patients, as some cases of menstrual dysfunction or spontaneous abortion were known.3,8 Familial cases with NSD1 mutations may be rare.

In conclusion, we have provided additional data of NSD1 mutations in SoS patients. These findings will contribute to further understanding of the function and structure of NSD1 and facilitate accurate diagnosis of SoS from other overgrowth syndromes.


We thank the patients and their parents for their participation to this study. We also express our gratitude to M Kazumi Miyazaki, Naoko Takaki, Naoko Yanai, and Yasuko Noguchi for their excellent technical assistance; and Drs A Orrico, Medical Genetics, Azienda Ospedaliera Senes, Italy; F Mari, Medical Genetics Unit, Department of Molecular Biology, University of Siena, Italy; Hidefumi Tonoki, Chitose Municipal Hospital, Japan; J Seidel at Kllinikum Der Friedrich Schiller University at Jena, Germany; Junji Nishimoto, Division of Pediatrics, Kinki Central Hospital, Japan; Kouji Inui, Department of Pediatrics, Osaka University Graduate School of Medicine, Japan; Mayumi Touyama, Okinawa Child Development Center, Japan; Reiko Kawamura, Hiroshima Jidou-Ryouiku-Shidou Center, Japan; Ryuji Fukuzawa, Cancer Genetics Laboratory Department of Biochemistry, University of Otago, New Zealand; Vazken M Der Kaloustian, Clinical Genetics Unit, Montreal Children’s Hospital, Canada; Yan-Ling Fei, Hospital For Sick Children, Canada, for their cooperation in sampling. This study was supported in part by CREST, Japan Science and Technology Corporation (JST).