Naohiro Kurotaki

Sotos syndrome and haploinsufficiency of NSD1: clinical features of intragenic mutations and submicroscopic deletions

Sotos syndrome (MIM 117550) is a congenital developmental disorder characterised by overgrowth and advanced bone age in infancy to early childhood, mental retardation, and various minor anomalies such as macrocephaly, prominent forehead, hypertelorism, downward slanting palpebral fissures, large ears, high and narrow palate, and large hands and feet.1,2 It is also frequently associated with brain, cardiovascular, and urinary anomalies3–6 and is occasionally accompanied by malignant lesions such as Wilms tumour and hepatocarcinoma.7,8 This condition has been classified as an autosomal dominant disorder, because several familial cases consistent with dominant inheritance have been described previously.9 Thus, sporadic cases accounting for most of the Sotos syndrome patients are assumed to be the result of de novo dominant mutations.nnWe have recently shown that Sotos syndrome is caused by haploinsufficiency of the gene for NSD1 (nuclear receptor binding Su-var, enhancer of zeste, and trithorax domain protein 1).10 NSD1 consists of 23 exons and encodes at least six functional domains possibly related to chromatin regulations (SET, PWWP-I, PWWP-II, PHD-I, PHD-II, and PHD-III), in addition to 10 putative nuclear localisation signals.11 It is expressed in several tissues including fetal/adult brain, kidney, skeletal muscle, spleen, and thymus11 and is likely to interact with nuclear receptors as a bifunctional transcriptional cofactor.12 In this paper, we report on clinical findings in Japanese patients with proven point mutations in NSD1 and those with submicroscopic deletions involving the entire NSD1 gene and discuss genotype-phenotype correlation.nnThis study consisted of five patients with heterozygous NSD1 point mutations and 21 patients with heterozygous submicroscopic deletions involving the entire NSD1 gene. The mutations were identified by direct sequencing of exons 2–23 and their flanking introns covering the whole coding region of NSD1 ,11 using genomic DNA extracted from peripheral leucocytes or …

Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene

NR-binding SET-domain-containing protein (NSD1) is a mouse nuclear protein containing su(var)3-9, enhancer-of-zeste, trithorax (SET), proline-tryptophan-tryptophan-proline (PWWP) and plant homeodomain protein (PHD)-finger domains (Huang et al., EMBO J. 17 (1998) 3398). This protein also has two other distinct nuclear receptor (NR)-interaction domains, called NID(-L) and NID(+L), and acts as both a NR corepressor and a coactivator by interacting directly with the ligand-binding domain of several NRs. Thus, NSD1 is a bifunctional, transcriptional, intermediary factor. We isolated the human homologue (NSD1) of the mouse NSD1 gene (Nsd1), mapped it to human chromosome 5q35, and characterized its genomic structure. NSD1 consists of at least 23 exons. Its cDNA is 8552 bp long, has an 8088 bp open reading frame, contains at least six functional domains (SET, PWWP-I, PWWP-II, PHD-I, PHD-II, and PHD-III) and ten putative nuclear localization signals, and encodes 2696 amino acids. NSD1 shows 86% identity with the mouse Nsd1 at the nucleotide level, and 83% at the amino acid level. NSD1 is expressed in the fetal/adult brain, kidney, skeletal muscle, spleen, and the thymus, and faintly in the lung. Two different transcripts (9.0 and 10.0 kb) were consistently observed in various fetal and adult tissues examined. These findings favor the character of NSD1 as a nucleus-localized, basic transcriptional factor and also a bifunctional transcriptional regulator, such as that of the mouse Nsd1. It remains to be investigated whether mutations of NSD1 lead to a specific phenotype in man.

Mutations in PRRT2 responsible for paroxysmal kinesigenic dyskinesias also cause benign familial infantile convulsions.

Paroxysmal kinesigenic dyskinesia (PKD (MIM128000)) is a neurological disorder characterized by recurrent attacks of involuntary movements. Benign familial infantile convulsion (BFIC) is also one of a neurological disorder characterized by clusters of epileptic seizures. The BFIC1 (MIM601764), BFIC2 (MIM605751) and BFIC4 (MIM612627) loci have been mapped to chromosome 19q, 16p and 1p, respectively, while BFIC3 (MIM607745) is caused by mutations in SCN2A on chromosome 2q24. Furthermore, patients with BFIC have been observed in a family concurrently with PKD. Both PKD and BFIC2 are heritable paroxysmal disorders and map to the same region on chromosome 16. Recently, the causative gene of PKD, the protein-rich transmembrane protein 2 (PRRT2), has been detected using whole-exome sequencing. We performed mutation analysis of PRRT2 by direct sequencing in 81 members of 17 families containing 15 PKD families and two BFIC families. Direct sequencing revealed that two mutations, c.649dupC and c.748C>T, were detected in all members of the PKD and BFIC families. Our results suggest that BFIC2 is caused by a truncated mutation that also causes PKD. Thus, PKD and BFIC2 are genetically identical and may cause convulsions and involuntary movements via a similar mechanism.

Preferential Paternal Origin of Microdeletions Caused by Prezygotic Chromosome or Chromatid Rearrangements in Sotos Syndrome

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.

Familial Sotos syndrome is caused by a novel 1 bp deletion of the NSD1 gene

Sotos syndrome (SS) or cerebral gigantism (OMIM *117550) is characterised by excessive growth, advanced bone age, typical facial gestalt, and developmental delay.1,2 In infancy growth is rapid, but settles down above the >97th centile in early childhood3,4 and tends to follow this during childhood. The adult height remains close to normal.4 The hands and feet are large. The facial gestalt is very characteristic during childhood with macrocephaly (>97th centile), frontal bossing, prognathism, hypertelorism, and antimongoloid slant of the palpebral fissures. With increasing age, the face gradually lengthens, the jaw becomes more prominent, and macrocephaly is no longer pronounced.5,6 Neurological features are variable and include hypotonia and delay in motor and language development, with a tendency for improvement with age.1,2,5–9 Familial SS is rare. Only 17 families have been reported, most of which show an autosomal dominant mode of inheritance.5,6,10–19nnRecent advances have shown a molecular genetic basis for sporadic SS. Initially, two SS patients carrying de novo balanced translocations suggested 5q35 as a possible locus for the SS gene.20,21 Subsequently, the NSD1 gene was isolated from the 5q35 breakpoint of the patient with t(5;8)(q35;q24.1) by positional cloning.22,23 NSD1 has an open reading frame of 8088 bp and consists of at least 23 exons (GenBank accession No AF395588). NSD1 is expressed in the fetal/adult brain, kidney, skeletal muscle, spleen, and the thymus, and faintly in the lung.22 The gene encodes 2696 amino acids with SET (su(var)3–9, enhancer-of-zeste, trithorax), PHD (plant homeodomain protein) finger, and PWWP (proline-tryptophan-tryptophan-proline) domains, all of which are possibly related to chromatin regulation, and possibly interact with nuclear receptors (Nrs).22 Among 42 sporadic SS patients examined by direct sequencing, altogether four de novo point mutations predicting truncation …

Phenotypic consequences of genetic variation at hemizygous alleles: Sotos syndrome is a contiguous gene syndrome incorporating coagulation factor twelve (FXII) deficiency.

Purpose: We tested the hypothesis that Sotos syndrome (SoS) due to the common deletion is a contiguous gene syndrome incorporating plasma coagulation factor twelve (FXII) deficiency. The relationship between FXII activity and the genotype at a functional polymorphism of the FXII gene was investigated.Methods: A total of 21 patients including those with the common deletion, smaller deletions, and point mutations, and four control individuals were analyzed. We examined FXII activity in patients and controls, and analyzed their FXII 46C/T genotype using direct DNA sequencing.Results: Among 10 common deletion patients, seven patients had lower FXII activity with the 46T allele of the FXII gene, whereas three patients had normal FXII activity with the 46C allele. Two patients with smaller deletions, whose FXII gene is not deleted had low FXII activity, but one patient with a smaller deletion had normal FXII. Four point mutation patients and controls all had FXII activities within the normal range.Conclusion: FXII activity in SoS patients with the common deletion is predominantly determined by the functional polymorphism of the remaining hemizygous FXII allele. Thus, Sotos syndrome is a contiguous gene syndrome incorporating coagulation factor twelve (FXII) deficiency.

Missense mutations in the DNA-binding/dimerization domain of NFIX cause Sotos-like features

Sotos syndrome is characterized by prenatal and postnatal overgrowth, characteristic craniofacial features and mental retardation. Haploinsufficiency of NSD1 causes Sotos syndrome. Recently, two microdeletions encompassing Nuclear Factor I-X (NFIX) and a nonsense mutation in NFIX have been found in three individuals with Sotos-like overgrowth features, suggesting possible involvements of NFIX abnormalities in Sotos-like features. Interestingly, seven frameshift and two splice site mutations in NFIX have also been found in nine individuals with Marshall–Smith syndrome. In this study, 48 individuals who were suspected as Sotos syndrome but showing no NSD1 abnormalities were examined for NFIX mutations by high-resolution melt analysis. We identified two heterozygous missense mutations in the DNA-binding/dimerization domain of the NFIX protein. Both mutations occurred at evolutionally conserved amino acids. The c.179T>C (p.Leu60Pro) mutation occurred de novo and the c.362G>C (p.Arg121Pro) mutation was inherited from possibly affected mother. Both mutations were absent in 250 healthy Japanese controls. Our study revealed that missense mutations in NFIX were able to cause Sotos-like features. Mutations in DNA-binding/dimerization domain of NFIX protein also suggest that the transcriptional regulation is abnormally fluctuated because of NFIX abnormalities. In individuals with Sotos-like features unrelated to NSD1 changes, genetic testing of NFIX should be considered.

Identification of eight novel NSD1 mutations in Sotos syndrome

In this study, we validated the spectrum of NSD1 intragenicmutations among 30 newly collected SoS patients.MATERIALS AND METHODSThe subjects studied included 13 Japanese and 17 non-Japanese patients with SoS. No patient with Weaver synromewas included in this study. The 17 non-Japanese casescomprised four Canadians including two Hutterite, threeeach of Brazilians, Germans, and Italians, and one each ofIsraeli-Arab, Israeli, Austrian, and Croatian. Three mainfeatures were considered at the clinical diagnosis: (a) typicalcraniofacial dysmorphology including macrocephaly, highanterior hairline, down slanting palpebral fissures, andprominent jaw, (b) developmental delay (intelligence quo-tient or development quotient ,80), and (c) history ofovergrowth (height and weight .+2 SD). Advanced bone agewas not evaluated, because sufficient data were not available.Adequate clinical information was available in 22 patients. Inthe other eight cases, only limited information was providedfor this study. All patients were referred to us aftermicrodeletions were ruled out by fluorescent in situhybridisation analysis using a P1 derived artificial chromo-some probe (RP11-118M12), as described previously.

Failure to confirm CNVs as of aetiological significance in twin pairs discordant for schizophrenia.

Copy number variations (CNVs) are common structural variations in the human genome that strongly affect genomic diversity and can play a role in the development of several diseases, including neurodevelopmental disorders. Recent reports indicate that monozygotic twins can show differential CNV profiles. We searched CNVs in monozygotic twins discordant for schizophrenia to identify susceptible loci for schizophrenia. Three pairs of monozygotic twins discordant for schizophrenia were subjected to analysis. Genomic DNA samples were extracted from peripheral blood lymphocytes. We adopted the Affymetrix Genome-Wide Human SNP (Single Nucleotide Polymorphism) Array 6.0 to detect copy number discordance using Partek Genomics Suite 6.5 beta. In three twin pairs, however, validations by quantitative PCR and DNA sequencing revealed that none of the regions had any discordance between the three twin pairs. Our results support the hypothesis that epigenetic changes or fluctuation in developmental process triggered by environmental factors mainly contribute to the pathogenesis of schizophrenia. Schizophrenia caused by strong genetics factors including copy number alteration or gene mutation may be a small subset of the clinical population.

Identification of Novel Schizophrenia Loci by Homozygosity Mapping Using DNA Microarray Analysis

The recent development of high-resolution DNA microarrays, in which hundreds of thousands of single nucleotide polymorphisms (SNPs) are genotyped, enables the rapid identification of susceptibility genes for complex diseases. Clusters of these SNPs may show runs of homozygosity (ROHs) that can be analyzed for association with disease. An analysis of patients whose parents were first cousins enables the search for autozygous segments in their offspring. Here, using the Affymetrix® Genome-Wide Human SNP Array 5.0 to determine ROHs, we genotyped 9 individuals with schizophrenia (SCZ) whose parents were first cousins. We identified overlapping ROHs on chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 19, 20, and 21 in at least 3 individuals. Only the locus on chromosome 5 has been reported previously. The ROHs on chromosome 5q23.3–q31.1 include the candidate genes histidine triad nucleotide binding protein 1 (HINT1) and acyl-CoA synthetase long-chain family member 6 (ACSL6). Other overlapping ROHs may contain novel rare recessive variants that affect SCZ specifically in our samples, given the highly heterozygous nature of SCZ. Analysis of patients whose parents are first cousins may provide new insights for the genetic analysis of psychiatric diseases.