American Journal of Medical Genetics Part C (Semin. Med. Genet.) 137C:24 –31 (2005) A R T I C L E NSD1 Mutations in Sotos Syndrome FRANCESCA FARAVELLI* Sotos syndrome is a genetic disorder characterized by a typical facial appearance, macrocephaly, accelerated growth, developmental delay, and a variable range of associated abnormalities. The NSD1 gene was recently found to be responsible for Sotos syndrome, and more than 150 patients with NSD1 alterations have been identified. A significant ethnic difference is found in the prevalence of different types of mutation, with a high percentage of microdeletions identified in Japanese Sotos syndrome patients and with intragenic mutations in most non-Japense patients. NSD1 aberrations are rather specific for Sotos syndrome, but have also been detected in patients lacking one or more major criteria of the disorder, namely overgrowth, macrocephaly, and advanced bone age. Thus, new diagnostic criteria should be considered. Studies have reported different frequencies of mutations versus non-mutations in Sotos syndrome, thus indicating allelic or locus hetereogeneity. Although some authors have suggested genotype/phenotype correlations, further studies are needed. ß 2005 Wiley-Liss, Inc. KEY WORDS: overgrowth syndromes; intragenic mutations; microdeletions; neoplasia; genotype/phenotype correlations; Weaver syndrome; Beckwith–Wiedemann syndrome INTRODUCTION Sotos syndrome (OMIM 117550) was described in 1964 in five patients with prenatal and postnatal overgrowth, characteristic facial appearance, advanced bone age, and developmental delay [Sotos et al., 1964], but most authors quote the report by Schlesinger  as the first case. Hundreds of cases were subsequently reported. Facial features are characterized by a high anterior hairline, frontal bossing, downslanting palpebral fissures, and prominent mandible [Sotos et al., 1964; Cole and Hughes, 1994; Allanson and Cole, 1996]. Additional features include neonatal hypotonia, poor feeding, jaundice, congenital heart defects, seizures, and scoliosis [Cole and Hughes, 1994; Opitz et al., 1998]. It remains to be determined Francesca Faravelli received her M.D. degree at the University of Genoa, Italy and trained in Medical Genetics in Genoa and in London, at the Institute of Child Health. She works as a clinical geneticist in the Department of Human Genetics of Galliera Hospital in Genoa. Her main field of interest is dysmorphology. *Correspondence to: Francesca Faravelli, SC Genetica Umana, Ospedale Galliera, Italy. DOI 10.1002/ajmg.c.30061 ß 2005 Wiley-Liss, Inc. to what extent Sotos syndrome is a cancer-predisposing syndrome [Hersh et al., 1992; Noreau et al., 1998; Cohen, 1999; Yule, 1999]. Phenotypic overlap with other overgrowth syndromes is known, particularly with Weaver syndrome, characterized by abnormal facial features, metaphyseal flaring of the femurs, camptodactyly, deep-set nails, hoarse low-pitched cry, and advanced bone age [Weaver et al., 1974; Cole et al., 1992; Opitz et al., 1998]. In 2002, the gene for Sotos syndrome was identified [Kurotaki et al., 2002] in a patient carrying an apparently balanced de novo translocation (5;8)(q35;q24.1) [Imaizumi et al., 2002]. Since then, microdeletions of NSD1 and intragenic mutations have been identified in more than 150 patients [Kurotaki et al., 2002; Douglas et al., 2003; Hoglund et al., 2003; Kamimura et al., 2003; Kurotaki et al., 2003; Nagai et al., 2003; Rio et al., 2003; Turkmen et al., 2003; Cecconi et al., 2005]. In 2002, the gene for Sotos syndrome was identified in a patient carrying an apparently balanced de novo translocation. Since then, microdeletions of NSD1 and intragenic mutations have been identified in more than 150 patients. NSD1 GENE AND PROTEIN The NSD1 gene was isolated and characterized in 2001 [Kurotaki et al., 2001]. It represents the human homolog of the mouse Nsd1 gene (Nuclear receptor-binding SET Domain containing protein 1). It was identified in 1998 in a search for factors mediating the transcriptional response induced by binding of ligands to nuclear receptors (NRs) [Huang et al., 1998]. It encodes the NSD1 protein, which belongs to an emerging family of proteins that include NSD2 (or MMSET and WHSCR1) [Chesi et al., 1998] and NSD3 [Angrand et al., 2001]. NSD2 is located in 4p16.3 in the Wolf–Hirschorn syndrome critical region of deletion [Stec et al., 2000]; NSD3 maps to 8p12. The three genes have similar functional domains. They have all been involved in human malignancies, suggesting a key role in ARTICLE controlling cell growth and differentiation. The NSD1 gene has been isolated recently in the context of a fusion transcript with the nucleoporin gene (NUP98) in a recurrent translocation in patients with de novo childhood acute myeloid leukemia [Jaju et al., 1999, 2001]. The NSD2 gene was disrupted in a t(4;14) translocation associated with lymphoid multiple myeloma [Chesi et al., 1998] and NSD3 is amplified in several tumor-derived cell lines and primary breast carcinomas [Angrand et al., 2001]. Recently, NSD3 has also been identified as a translocation partner of NUP98 in acute myeloid leukemia [Rosati et al., 2002]. NSD1 has an open reading frame of 8,088 base pairs, consists of 23 exons, and is translated into a 2,696 amino acid protein [Kurotaki et al., 2001]. It contains multiple functional domains, including the SET domain (SU[VAR]3–9, Enhancer of Zeste, Trithorax), initially identified in Drosophila genes involved in chromatin-mediated regulation during development [Tschiersch et al., 1994; reviewed in Jenuwein, 2001]. It also contains a SETassociated Cys-rich (SAC) domain adjacent to the SET domain, typical of proteins functioning as histone-methyltransferases (HMTases) [Rea et al., 2000]. Other functional domains are represented by five plant homeodomain (PHD): zinc finger-like motifs interacting with chromatin and two PWWP domains thought to be involved in protein–protein interaction [Aasland et al., 1995; Stec et al., 2000]. Finally, the NSD1 gene contains two distinct NR interaction domains (NID): NIDL and NIDþL that exhibit binding properties of NIDs found in NR corepressors and coactivators [Huang et al., 1998]. This suggests that NSD1 may function as a bifunctional transcriptional regulator that can activate or repress transcription in response to ligand binding [Huang et al., 1998]. The expression pattern of NSD1 revealed the presence of two different transcripts (9.0 kb and 10.0 kb) in various fetal and adult tissues, including brain, kidney, skeletal muscle, spleen, thymus, and lung. To gain further insights into its biological function, Nsd1 deficient mice have recently been AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) generated by gene disruption [Rayasam et al., 2003]. Homozygous mutant embryos fail to complete gastrulation, indicating that NSD1 is essential for early post-implantation development. By contrast, heterozygous mutant miceþ/ are viable, fertile, and display early normal growth rate [Rayasam et al., 2003], indicating that the Sotos phenotype in mice may be milder than in humans. Furthermore, the SET domain represents a novel HMTase domain with a specific histone methyltransferase activity [Rayasam et al., 2003]. NSD1 ABERRATIONS IN SOTOS SYNDROME Kurotaki et al.  cloned the 5q35 breakpoint of a translocation (5;8) in a Sotos syndrome patient and demonstrated that the NSD1 gene was disrupted in this rearrangement. The same authors identified microdeletions encompassing NSD1 as the major cause of Sotos syndrome in Japanese patients. This finding was subsequently confirmed in a larger series of Japanese patients [Kurotaki et al., 2003], but not in European patients who tended to harbor intragenic mutations [Douglas et al., 2003; Kurotaki et al., 2003; Rio et al., 2003; Turkmen et al., 2003; Cecconi et al., 2005]. Table I shows the frequency of NSD1 aberrations in reported patients and clinical criteria used for their selection. Although most authors refer to the criteria suggested by Cole and Hughes , that is a typical facial gestalt, overgrowth, macrocephaly, and advanced bone age, some differences are found in published series, and this probably explains the variability in the overall detection rate of mutations in Sotos syndrome (35%–90%). In particular, most reports distinguish a class of typical Sotos syndrome patients and one of Sotos-like patients. The second group refers to individuals lacking one or more major criteria including, in some instances, the typical facial gestalt. In this group, a few patients with mutations have been identified and this can be explained by the subsequent observation that a typical facial gestalt represents a 25 constant criterion in individuals with NSD1 aberrations (see section titled Clinical Features in Patients With NSD1 Alterations). A variable proportion of patients with a clinical diagnosis of Sotos syndrome do not show alterations in NSD1. This is likely due, at least in part, to allelic heterogeneity with some patients having underlying aberrations undetectable by means of currently used screening techniques. A partial deletion of the gene A variable proportion of patients with a clinical diagnosis of Sotos syndrome do not show alterations in NSD1. This is likely due, at least in part, to allelic heterogeneity with some patients having underlying aberrations undetectable by means of currently used screening techniques. detected by use of quantitative multiplex PCR of short fluorescent fragments (QMPSF) has recently been described in one such patient [Drouin-Garraud et al., 2003]. However, it cannot be excluded that locus heterogeneity exists in this disorder. Baujat et al.  described anomalies of the 11p15 region in two patients with a clinical diagnosis of Sotos syndrome, though neither was typical of the condition. The first patients, with documented paternal uniparental disomy of the 11p15 region, had previously been published as a Sotos-like phenotype with absence of advanced bone age [Amiel et al., 2002, their case 3]. It remains to be determined whether some cases of Sotos syndrome are allelic to Beckwith–Wiedemann syndrome. The NSD2 and NSD3 genes were recently tested as potential candidates for NSD1-negative Sotos syndrome patients based on strong sequence similarities to the NSD1 gene. No mutations or 26 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE TABLE I. Reported Frequencies of NSD1 Aberrations in Sotos Syndrome Patients Intragenic mutations Typical SoS Kurotaki et al.  Kurotaki et al. a Japanese patients Non-Japanese patients Douglas et al. b Rio et al. c Turkmen et al. d Cecconi et al. e SoS-like 4/38 (10%) 11/95 5/17 26/37 14/23 19/21 16/24 (12%) (29%) (70%) (61%) (90%) (66%) 3/13 (23%) 2/10 (20%) 0/9 Deletions Typical SoS SoS-like Overall frequency of NSD1 aberrations in typical SoS 20/30 (67%) (77%) 49/95 (52%) 1/17 (6%) 2/37 (5%) 2/23 (9%) — 1/24 (4%) 60/95 (63%) 6/17 (35%) 28/37 (76%) 16/23 (69%) 19/21 (90%) 17/24 (70%) 1/13 (7%) 4/10 (4%) 0/9 SoS: Sotos syndrome. Clinical criteria adopted as follows: a Typical Sotos: typical facial gestalt, mental retardation, and overgrowth. b Typical Sotos: presence of four criteria of: typical facial gestalt, height >97th centile, OFC >97th centile, bone age >90th centile, developmental delay, congenital anomalies, or malformations; Sotos-like: some atypical characteristics, primarily concerning facial features. c Typical Sotos: presence of four criteria: typical facial gestalt, overgrowth >97th centile, OFC >97th centile, advanced bone age; Sotos-like: lacking bone age and overgrowth. d Typical Sotos: presence of four criteria of five: typical facial gestalt, overgrowth >97th centile, OFC >97th centile, advanced bone age, developmental delay. e Typical Sotos: presence of four of five major criteria: typical facial gestalt, overgrowth >97th centile, OFC >97th centile, advanced bone age, developmental delay; Sotos-like: some atypical characteristics, primarily concerning facial features. This estimate is as reported from the authors. deletions were identified in 78 patients with overgrowth [Douglas et al., 2004]. NSD1 MICRODELETIONS Microdeletions encompassing the NSD1 gene have been reported in approximately 50% of Japanese patients and in less than 10% of non-Japanese patients [Table II]. Preferential paternal Microdeletions encompassing the NSD1 gene have been reported in approximately 50% of Japanese patients and in less than 10% of non-Japanese patients. origin of microdeletions has been noted [Douglas et al., 2003, Miyake et al., 2003, Rio et al., 2003], as for other de novo mutations and structural chromosomal abnormalities [Chandley, 1991]. A common-sized 2 Mb deletion is very frequent in Japanese patients and two complex low-copy repeats (LCRs) have been identified at both common deletion breakpoints [Kurotaki et al., 2003]. The size, structure, orientation, and extent of sequence identity of these LCRs have been fully characterized and a non-allelic homologous recombination (NAHR) between directly oriented LCRs subunits have been demonstrated to mediate the common deletion [Kurotaki et al., 2005; Visser et al., 2005]. Investigating 33 non-Japanese NSD1 microdeletion cases, TattonBrown et al. [in press] confirmed that although the majority of cases have a same-sized deletion, a high proportion of non-recurrent microdeletions of variable size exist [Douglas et al., 2003; Kurotaki et al., 2003] and suggested possible mechanistic difference in the generation of the microdeletion among different populations. The reason for these discrepancies and for the observed difference in frequency of the common deletion remains to be determined. To date, no instances of familial transmis- sion of an NSD1 microdeletion have been reported. NSD1 INTRAGENIC MUTATIONS More than 100 distinct intragenic mutations in NSD1 have been identified [Kurotaki et al., 2002; Douglas et al., 2003; Nagai et al., 2003; Rio et al., 2003; Turkmen et al., 2003; Cecconi et al., 2005]. No mutational hot spots are present in the gene and mutations are widespread through the 23 exons with a few recurring changes described in small numbers of unrelated patients. Most mutations have a truncating effect on the protein. A minority of missense mutations affecting functional domains have been identified. Finally, all mutations arose de novo, with the exception of three familial cases with documented transmission of the phenotype from an affected parent [Douglas et al., 2003; Hoglund et al., 2003; Kurotaki et al., 2003]. Table II summarizes all reported intragenic mutations. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) TABLE II. Reported NSD1 Intragenic Mutations Mutation Splice-site IVS8-2A ! G IVS15-1G ! C IVS15-1G ! T IVS16-2delA IVS19-2A ! G 6151þ1G ! A Deletion 896delC 1171delC 1727delA 1807delT 2053-2057del 2363delG 2386delGAAA 2432delG 2576delAT 2760delTAGG 3141delC 3160delA 3197delC 3273delT 3355delC 3383delCT 3536delA 3541delGAAA 3708delTTGT 3841delC 3844delTTGGAinsGATC 4139delAAGTCinsCTG 4806delTGTTAAA 4883delT 4895delG 5341delC 6001delC 6302delA 6532delTGCCCCAGC Nonsense 1130G ! A 1310C ! G 1318C ! T 1492C ! T 1492C ! T 1810C ! T 1828C ! T 2323C ! T 3063T !A 3091C ! T 3214C ! T 3886A !T Protein change (where reported) Exon Mutation analysis in parents Reference Skip ex9/ frameshift — Skip ex16/frameshift — Skip ex20/frameshift Skip ex20 Int 8 Int 15 Int 15 Int 16 Int 19 Int 20 Neg — — Neg [m] Neg Neg 5 2 5 2 5 1 Frameshift Q391fsX418 N576fsX598 Frameshift Frameshift Frameshift E796fsX805 Frameshift H859fsX873 Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift S1128fsX1129 Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift M1628fsX1641 Frameshift Frameshift L2001fsX2001 K2101fsX2149 2178-2180delCPS 2 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 7 13 13 13 16 19 22 23 Pos [f] Neg — — — Neg Pos [m] — — Neg Neg [m] — Neg — Neg — Neg — Neg Neg Neg Neg Neg Neg [m] — Neg — Neg Familial 8 2 2 6 6 3 4 4 2 3 5 5 3 6 3 2 1, 5 5 3 3 3 3 5 2 5 4 2 2 5 W377X S437X R440X R498X R498X R604X Q610X Q775X C1021X R1031X R1072X K1296X 4 5 5 5 5 5 5 5 5 5 5 6 — Neg Neg — — Neg Neg Neg Neg Neg Neg Neg 6 1 3 2 5 2 4 2 3 3, 5 3 6 (Continued ) 27 28 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE TABLE II. (Continued ) Mutation 3958C ! T 3964C ! T 4411C ! T 4417C ! T 4885C ! T 5194G ! T 5229G ! A 5332C ! T 5431C ! T 5611A !T 5861G ! A 6013C ! T Insertion 2807-8insA 3549-50insT 4160insC 4390delATATins8 4769insT 5008-9insG 5398insT 5744-5insT 5998insT 6431-2ins17 6450-1insC Missense 4411C ! G 4847A !T 4910T ! C 5022C ! G 5060T !A 5178T ! C 5179G ! C 5375G ! T 5386G ! T 5684G ! A 5773T ! C 5864G ! A 5951G ! A 5990A ! G 6014G ! A 6049C ! T 6050G ! A 6429C ! G 6499T ! C 6548G ! C 6680C ! T Protein change (where reported) Exon Mutation analysis in parents Reference R1320X R1322X R1471X R1473X Q1629X E1732X W1743X R1778X R1811X K1871X W1954X R2005X 7 7 10 10 13 15 15 16 16 17 18 20 Neg Neg Neg [m] Neg — — — — Neg Neg Neg Neg 7 6 2 2 5 5 6 2 3 5 2 5 Y936fsX936 E1184fsX1184 Frameshift Frameshift Frameshift A1670fsX1672 Frameshift M1915fsX1919 Frameshift A2144fsX2155 K2151fsX2165 5 5 7 10 13 14 16 18 19 22 22 — — Neg Neg Neg — Neg Neg Neg Neg Neg [m] 2 2 5 3 6 2 5 2 1 2 2 R1471G H1616L L1637P C1674W I1687N P1726L A1725P G1792V V1796F C1895Y C1925R G1955D R1984Q Y1997C R2005Q R2017W R2017Q H2143E C2167R C2183S P2227L 10 13 13 14 14 15 15 16 16 18 18 18 19 19 20 20 20 22 23 23 23 — Neg Neg — Neg — — Neg Neg Neg Neg [m] Neg Neg Neg Neg Neg Neg Neg — Neg — 4 2 2 2 3 4 4 2 5 4 2 3 3 3 2 3 2 2 4 2 4 Neg, negative; Neg [m], only the mother examined; Neg [f], only the father examined; Pos [m], positive in the mother; Pos [f], positive in the father; —, not examined. References: 1, Kurotaki et al. ; 2, Douglas et al. ; 3, Rio et al. ; 4, Kurotaki et al. ; 5, Turkmen et al. ; 6, Kamimura et al. ; 7, Nagai et al. ; 8, Hoglund et al. . ARTICLE CLINICAL FEATURES IN PATIENTS WITH NSD1 ALTERATIONS Detailed clinical information is available for a subset of 86 patients with NSD1 alterations (58 patients with intragenic mutations and 28 with deletions) [Hoglund et al., 2003; Nagai et al., 2003; Rio et al., 2003; Turkmen et al., 2003; Cecconi et al., 2005]. AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) series. However, a substantial proportion of patients with confirmed Sotos syndrome are described as having a normal or moderately advanced bone age (25%, n ¼ 69). Patients with NSD1 aberrations and reduced bone age have also been identified [Tatton-Brown and Rahman, 2004]. Because of this data, advanced bone age is not considered a mandatory diagnostic criterion. DEVELOPMENTAL DELAY CRANIOFACIAL FEATURES In Sotos syndrome, the most striking facial feature is the overall gestalt [Cole and Hughes, 1994], which includes high and bossed forehead, downslanting palpebral fissures, highly arched palate, and a small pointed chin, which becomes more prominent with age [Allanson and Cole, 1996; Tatton-Brown and Rahman, 2004]. Clinical analysis of reported patients with a confirmed NSD1 abnormality suggests that the facial phenotype is the most constant feature. OVERGROWTH AND MACROCEPHALY Overgrowth and macrocephaly are major criteria for the diagnosis of Sotos syndrome [Cole and Hughes, 1994]. Most authors define overgrowth in Sotos syndrome as height >97th centile or þ2 SD. A significant proportion of patients with NSD1 alterations do not satisfy this criterion. Some authors postulated that overgrowth could be less evident in patients with a microdeletion. Although available data are partially consistent with this hypothesis, such as absent overgrowth in 92% (n ¼ 58) with intragenic mutations and 21% (n ¼ 28) with microdeletions, analysis of a larger series is needed. Macrocephaly (>97th centile) is absent in about 7% (n ¼ 85). Information about parental height and head circumferences is rarely available. ADVANCED BONE AGE A unified method of bone age assessment and criteria for the definition of advanced bone age are absent in reported A formal assessment of the degree of intellectual impairment has rarely been performed, although all patients are described as having some degree of mental retardation/developmental delay. ASSOCIATED CONGENITAL ANOMALIES Congenital heart defects and urogenital abnormalities are relatively frequent, and the type of NSD1 alteration may influence their frequency. Congenital heart defects were reported in 8.7% (n ¼ 23) of patients with intragenic mutations [Rio et al., 2003] and in 55.5% (n ¼ 27) of patients with microdeletions [Nagai et al., 2003; Rio et al., 2003]. The most frequent defect is patent ductus arteriosus with or without atrial septal defect. Variability in the Congenital heart defects were reported in 8.7% of patients with intragenic mutations and in 55.5% of patients with microdeletions. The most frequent defect is patent ductus arteriosus with or without atrial septal defect. prevalence of congenital heart defects has been noted in different populations (40%–50% in Japanese and 8%–10% in non-Japanese patients) [Kaneko et al., 1987; Noreau et al., 1998; Tsukahara et al., 1999]. Some authors have postulated that a second gene in the deleted 29 region might be involved in the pathogenesis of structural heart abnormalities [Visser and Matsumoto, 2003]. However, recent data on patients with intragenic mutations and a high prevalence of congenital heart defects (7/16 patients) [Cecconi et al., 2005] do not support this hypothesis. Haploinsufficiency of the 5q31qter region is associated with a high frequency of congenital heart defects [Kleczkowska et al., 1993; Stratton et al., 1994; Pauli et al., 1999]. The NKX2.5 gene, whose mutations are associated with congenital heart defects and atrioventricular conduction defects, is localized to this region [Schott et al., 1998; Benson et al., 1999] and has been indicated as being responsible for the cardiac phenotype [Schafer et al., 2001]; most of these deletions involve NSD1, which could itself contribute to the presence of congenital heart defects in these patients. NEOPLASIA Two patients with confirmed Sotos and neuroblastoma have been reported, one with a 5q35 microdeletion and another with an intragenic mutation [Nagai et al., 2003; Turkmen et al., 2003]. A third patient with ganglioglioma and a microdeletion has been noted [Deardorff et al., 2004]. Three sacroccygeal teratomas, one ganglioneuroma, one neuroblastoma, and one case of acute lymphocytic leukemia have occurred in childhood, and one small cell lung cancer at 22 years of age occurred in a series of NSD1 positive cases [Tatton-Brown and Rahman, 2004]. These data support the association of Sotos syndrome and malignancy. A larger series is necessary to establish a more accurate tumor prevalence. GENOTYPE–PHENOTYPE CORRELATION IN SOTOS SYNDROME As discussed above, some preliminary suggestions have been made about genotype–phenotype correlation in Sotos syndrome. The facial features have been suggested to be coarser in microdeleted patients, although this is highly 30 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) subjective [Douglas et al., 2003]. A more severe degree of mental retardation but less frequent and pronounced overgrowth has been recorded in microdeleted patients [Douglas et al., 2003; Nagai et al., 2003; Rio et al., 2003]. A higher incidence of congenital heart defects and urologic anomalies has been observed in patients with microdeletions [Nagai et al., 2003; Rio et al., 2003], although these are also reported with variable frequency in patients with intragenic mutations [Rio et al., 2003; Cecconi et al., 2005]. Careful clinical evaluation of larger series of patients is needed to verify these observations. NSD1 ABERRATIONS IN OTHER OVERGROWTH PHENOTYPES NSD1 aberrations were demonstrated in one-third of investigated patients with a diagnosis of Weaver syndrome (6/18) [Douglas et al., 2003; Rio et al., 2003; Turkmen et al., 2003]. Reported features of mutated patients, when available, included deep-set nails (three cases), camptodactyly (three cases), small chin with deep horizontal skin crease (two cases), and metaphyseal widening (two cases). Further data are needed to verify if both disorders are actually allelic or if some heterogeneity exists in the pathogenesis of Weaver syndrome. No NSD1 alterations were demonstrated in nearly 50 patients with non-specific overgrowth phenotype [Douglas et al., 2003; Rio et al., 2003; Cecconi et al., 2005]. NSD1 mutations have been reported in two patients classified clinically as having Beckwith–Wiedemann syndrome [Baujat et al., 2004]. OPEN ISSUES AND FUTURE PROSPECTS Available data leave many issues about Sotos syndrome and the NSD1 gene unanswered. To date, the reason for differences in mutation type among different populations is undetermined. In all ethnic groups, a proportion of patients with a clinical diagnosis of Sotos syndrome do not have detectable NSD1 aberrations and it is not known if this is due to allelic or locus heterogeneity. A definitive genotype–phenotype correlation awaits further studies. In the future, it will be important to better understand the functional role of the NSD1 gene and pathogenetic mechanisms in Sotos syndrome, to identify other factors that mediate phenotypic variability in the presence of similar genotypes, and to determine whether the NSD1 gene is a major contributor to other overgrowth phenotypes, such as Weaver syndrome. A large series of patients with confirmed aberrations in the NSD1 gene will allow a better estimate of the neoplastic risk and possibly an understanding of other factors that may influence this risk. ACKNOWLEDGMENTS I thank Nazneen Rahman for helpful comments and for collaboration. REFERENCES Aasland R, Gibson TJ, Stewart AF. 1995. 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