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• W2054408230 abstract "•The APC2 gene is homozygously mutated in two siblings with Sotos syndrome features•APC2 is downstream of NSD1, the main causative gene of Sotos syndrome•The Apc2-deficient mouse is a model for Sotos syndrome Sotos syndrome, characterized by intellectual disability and characteristic facial features, is caused by haploinsufficiency in the NSD1 gene. We conducted an etiological study on two siblings with Sotos features without mutations in NSD1 and detected a homozygous frameshift mutation in the APC2 gene by whole-exome sequencing, which resulted in the loss of function of cytoskeletal regulation in neurons. Apc2-deficient (Apc2−/−) mice exhibited impaired learning and memory abilities along with an abnormal head shape. Endogenous Apc2 expression was downregulated by the knockdown of Nsd1, indicating that APC2 is a downstream effector of NSD1 in neurons. Nsd1 knockdown in embryonic mouse brains impaired the migration and laminar positioning of cortical neurons, as observed in Apc2−/− mice, and this defect was rescued by the forced expression of Apc2. Thus, APC2 is a crucial target of NSD1, which provides an explanation for the intellectual disability associated with Sotos syndrome. Sotos syndrome, characterized by intellectual disability and characteristic facial features, is caused by haploinsufficiency in the NSD1 gene. We conducted an etiological study on two siblings with Sotos features without mutations in NSD1 and detected a homozygous frameshift mutation in the APC2 gene by whole-exome sequencing, which resulted in the loss of function of cytoskeletal regulation in neurons. Apc2-deficient (Apc2−/−) mice exhibited impaired learning and memory abilities along with an abnormal head shape. Endogenous Apc2 expression was downregulated by the knockdown of Nsd1, indicating that APC2 is a downstream effector of NSD1 in neurons. Nsd1 knockdown in embryonic mouse brains impaired the migration and laminar positioning of cortical neurons, as observed in Apc2−/− mice, and this defect was rescued by the forced expression of Apc2. Thus, APC2 is a crucial target of NSD1, which provides an explanation for the intellectual disability associated with Sotos syndrome. Overgrowth syndromes are a heterogeneous group of disorders defined by the generalized or localized excess growth of a body part (Visser et al., 2009Visser R. Kant S.G. Wit J.M. Breuning M.H. Overgrowth syndromes:from classical to new.Pediatr. Endocrinol. Rev. 2009; 6: 375-394PubMed Google Scholar). One prominent overgrowth syndrome is Sotos syndrome (OMIM #117550; also known as cerebral gigantism, with an estimated incidence of 1/14,000 live births), which is characterized by varying degrees of mental retardation and a combination of typical facial features (i.e., a prominent forehead with a receding hairline, downslanting palpebral fissures, and a pointed chin) and large head circumference (Cole and Hughes, 1994Cole T.R. Hughes H.E. Sotos syndrome: a study of the diagnostic criteria and natural history.J. Med. Genet. 1994; 31: 20-32Crossref PubMed Scopus (217) Google Scholar, Sotos et al., 1964Sotos J.F. Dodge P.R. Muirhead D. Crawford J.D. Talbot N.B. Cerebral gigantism in childhood. A syndrome of excessively rapid growth and acromegalic features and a nonprogressive neurologic disorder.N. Engl. J. Med. 1964; 271: 109-116Crossref PubMed Scopus (276) Google Scholar, Tatton-Brown et al., 2012Tatton-Brown K. Cole T.R.P. Rahman N. Sotos Syndrome. GeneReviews, 2012Google Scholar). Additional features may include hypotonia, advanced bone age, seizures, prognathia, cardiac and renal anomalies, and scoliosis. The NSD1 (Nuclear receptor-binding Set Domain containing 1) gene, which encodes a histone methyltransferase that has been implicated in transcriptional regulation, was previously reported to be deleted or mutated in ∼90% of Sotos syndrome cases (Baujat and Cormier-Daire, 2007Baujat G. Cormier-Daire V. Sotos syndrome.Orphanet J. Rare Dis. 2007; 2: 36Crossref PubMed Scopus (94) Google Scholar, Douglas et al., 2003Douglas J. Hanks S. Temple I.K. Davies S. Murray A. Upadhyaya M. Tomkins S. Hughes H.E. Cole T.R. Rahman N. NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes.Am. J. Hum. Genet. 2003; 72: 132-143Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Kurotaki et al., 2002Kurotaki N. Imaizumi K. Harada N. Masuno M. Kondoh T. Nagai T. Ohashi H. Naritomi K. Tsukahara M. Makita Y. et al.Haploinsufficiency of NSD1 causes Sotos syndrome.Nat. Genet. 2002; 30: 365-366Crossref PubMed Scopus (473) Google Scholar). NSD1 is considered to regulate the expression of a battery of genes required for the normal development of several tissues; however, the downstream effector molecules encoded by these genes and the mechanisms that cause individual Sotos features currently remain unknown. We recently demonstrated that APC2 (Adenomatous polyposis coli 2), an APC-like protein, is crucially involved in brain development through its regulation of neuronal migration and axon guidance (Shintani et al., 2009Shintani T. Ihara M. Tani S. Sakuraba J. Sakuta H. Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability.J. Neurosci. 2009; 29: 11628-11640Crossref PubMed Scopus (32) Google Scholar, Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). APC is a microtubule plus-end tracking protein (+TIP) and is also known as a tumor suppressor (Midgley et al., 1997Midgley C.A. White S. Howitt R. Save V. Dunlop M.G. Hall P.A. Lane D.P. Wyllie A.H. Bubb V.J. APC expression in normal human tissues.J. Pathol. 1997; 181: 426-433Crossref PubMed Scopus (85) Google Scholar, Mimori-Kiyosue et al., 2000Mimori-Kiyosue Y. Shiina N. Tsukita S. Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells.J. Cell Biol. 2000; 148: 505-518Crossref PubMed Scopus (249) Google Scholar). APC is broadly expressed in most mammalian tissues (Midgley et al., 1997Midgley C.A. White S. Howitt R. Save V. Dunlop M.G. Hall P.A. Lane D.P. Wyllie A.H. Bubb V.J. APC expression in normal human tissues.J. Pathol. 1997; 181: 426-433Crossref PubMed Scopus (85) Google Scholar), whereas APC2 is preferentially expressed in postmitotic neurons throughout development (Shintani et al., 2009Shintani T. Ihara M. Tani S. Sakuraba J. Sakuta H. Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability.J. Neurosci. 2009; 29: 11628-11640Crossref PubMed Scopus (32) Google Scholar, Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). APC2 has been shown to co-localize with and stabilize microtubules (Shintani et al., 2009Shintani T. Ihara M. Tani S. Sakuraba J. Sakuta H. Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability.J. Neurosci. 2009; 29: 11628-11640Crossref PubMed Scopus (32) Google Scholar). It is also distributed along actin fibers and influences their dynamics by regulating the activation of Rho family GTPases (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). Severe layering defects were detected in some brain regions of Apc2-deficient (Apc2−/−) mice due to dysregulated neuronal migration, and Apc2−/− neurons were shown to be defective in directional migration in response to extracellular guidance molecules in vitro (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). APC2 also plays a role in axonal path-finding by regulating the responsiveness of axonal growth cones to guidance molecules (Shintani et al., 2009Shintani T. Ihara M. Tani S. Sakuraba J. Sakuta H. Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability.J. Neurosci. 2009; 29: 11628-11640Crossref PubMed Scopus (32) Google Scholar). Therefore, APC2 functions in brain development through cytoskeletal regulation in neurons in response to endogenous extracellular signals. We previously speculated that loss of APC2 function may be involved in the etiology of human genetic neurological disorders (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). Here, we performed an etiological study on two siblings with Sotos-like overgrowth features: intellectual disability and relative macrocephaly with a long face and prominent chin and nose. Whole-exome sequencing (WES) of these patients revealed a homozygous frameshift mutation in the APC2 gene, but no mutations in NSD1 or other known susceptibility genes. Cell-based functional assays indicated that the mutant APC2 of these patients was a functionally null protein. Apc2−/− mice exhibited significantly impaired learning and memory abilities together with an abnormal brain structure and head shape, indicating a high degree of concordance between the phenotypes observed in human patients and knockout mice. Expression of APC2 was also revealed to be under the control of NSD1. Impaired migration of cortical neurons was observed when Nsd1 was knocked down in the embryonic brain, as in the Apc2−/− brain, and this defect was rescued by the forced expression of Apc2. The results of the present study provide a detailed mechanism for the intellectual disability associated with Sotos syndrome. The patients’ parents were healthy first cousins of Egyptian descent and only had two affected children: a girl (patient 1, aged 10 years) and boy (patient 2, aged 8 years). Both affected siblings were born at term after an uneventful pregnancy, and early development was within normal limits. However, they both had a severe receptive and expressive language disorder and learning difficulties as well as hyperactive behavior. Their intelligence quotients were 60 and 56 for the girl and boy, respectively. The girl had an occipital frontal circumference (OFC) of 53 cm (the 98th percentile), weight of 20.4 kg, and height of 117 cm (both in the 25th percentile) at 7 years (89 months) of age, and the boy had an OFC of 50 cm (in the 98th percentile), weight of 23.3 kg (50th percentile), and height of 120 cm (90th percentile) at 5 years (64 months) of age. Sotos syndrome was suspected based on the intellectual disability and physical findings of relative macrocephaly with a long face and prominent chin and nose (Figure 1A). These patients did not have any other disorders such as advanced bone age, hypotonia, seizures, or an autistic phenotype. In view of the parents’ consanguinity, the most likely mode of inheritance in this family was considered to be autosomal recessive. Array-based comparative genomic hybridization identified no copy-number changes in any known genes relevant to the clinical features of our patients (data not shown). We subsequently performed WES on the genomic DNA of the two patients as described previously (Fahiminiya et al., 2014Fahiminiya S. Almuriekhi M. Nawaz Z. Staffa A. Lepage P. Ali R. Hashim L. Schwartzentruber J. Abu Khadija K. Zaineddin S. et al.Whole exome sequencing unravels disease-causing genes in consanguineous families in Qatar.Clin. Genet. 2014; 86: 134-141Crossref PubMed Scopus (70) Google Scholar, McDonald-McGinn et al., 2013McDonald-McGinn D.M. Fahiminiya S. Revil T. Nowakowska B.A. Suhl J. Bailey A. Mlynarski E. Lynch D.R. Yan A.C. Bilaniuk L.T. et al.Hemizygous mutations in SNAP29 unmask autosomal recessive conditions and contribute to atypical findings in patients with 22q11.2DS.J. Med. Genet. 2013; 50: 80-90Crossref PubMed Scopus (94) Google Scholar). We did not detect any rare sequence variations within the reported genes associated with Sotos syndrome (NSD1) or other overgrowth syndromes (NFIX, EZH2, PTEN, DNMT3A, and SETD2). However, a homozygous single-nucleotide duplication of C was detected in exon 15 (c.5199 dup) of the APC2 gene (NM_005883) in both patients, which was located within a 3.2-Mb overlapping region of homozygosity (Figures 1B and 1C; Table S1). This duplication resulted in a frameshift in the open reading frame (ORF) of the APC2 gene, in which 570 amino acid residues in the C terminus were replaced with 418 aberrant residues (p.[Lys1734Glnfs∗418]). Subsequent sequencing analyses revealed that their parents were heterozygous for the APC2 mutation, consistent with an autosomal-recessive mode of inheritance (Figure 1C). To identify functional alterations in the mutated APC2 protein encoded by the patients’ genomes at the cellular level, we constructed three human APC2 constructs: WT hAPC2, Mut hAPC2, and ΔC hAPC2 (Figure 2A). We expressed them in Neuro2a cells that were endogenously devoid of detectable levels of APC2 proteins (Figure S1). Neuro2a is generally used to study cytoskeletal regulation in neurons. N-terminally tagged wild-type (WT) and mutant APC2 proteins were expressed with the expected molecular sizes in Neuro2a cells (Figure S2A). We first verified that N-terminal tagging did not alter the subcellular distribution or microtubule-stabilizing activities of WT hAPC2 (Figure S2). As expected, the WT hAPC2 proteins were co-localized with microtubules (Figure S2B). We then estimated microtubule stabilization activities by using the microtubule-destabilizing agent nocodazole. The WT hAPC2 exhibited the ability to stabilize microtubules (Figure S2C), as previously reported for WT APC2 proteins with or without C-terminal tags (Shintani et al., 2009Shintani T. Ihara M. Tani S. Sakuraba J. Sakuta H. Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability.J. Neurosci. 2009; 29: 11628-11640Crossref PubMed Scopus (32) Google Scholar, Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). Of note, neither of the APC2 mutant proteins (Mut hAPC2 and ΔC hAPC2) was distributed along the microtubules (Figure 2B), and they exhibited a large punctate pattern (see below). Furthermore, when we estimated the amount of acetylated tubulins (the stabilized form of microtubules), we found that the two APC2 mutants had lost the microtubule stabilization activities of WT APC2 (Figure 2C). APC2 has been shown to co-localize with F-actin and stimulate the activity of the Rho family GTPase, Rac1 (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). The WT hAPC2 was co-localized with F-actins in Neuro2a cells, whereas the APC2 mutants were not (Figure 3A). The amount of active Rac1 was ∼1.4-fold higher in WT hAPC2-expressing cells than in mCherry-expressing control cells (Figure 3B), as previously reported (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). In contrast, the expression of Mut hAPC2 or ΔC hAPC2 did not significantly upregulate the level of active Rac1 (Figure 3B), indicating that both mutants were defective for increasing the activity of Rac1. It should be noted that Mut hAPC2 and ΔC hAPC2 had no dominant-negative activities on the WT hAPC2 protein (Figure S3). The mislocalization of mutant APC2 proteins was also examined in cortical neurons derived from Apc2−/− mice, which may reflect more in vivo conditions than Neuro2a. In primary cultured cortical neurons, WT hAPC2 proteins were distributed along microtubules throughout the neurites, growth cones, and cell bodies, whereas both of the mutant forms (Mut hAPC2 and ΔC hAPC2) were prominently concentrated in the cell bodies (Figures 3C and 3D), suggesting that the C-terminal region (570 amino acids) of WT hAPC2 was indispensable for its proper distribution in endogenous neurons. Unlike the case with Neuro2a cells (see above, Figure 2B), no characteristic punctate (vesicular) signals were observed in primary cultured cortical neurons (Figure 3D). Therefore, the punctate distribution of Mut APC2 in Neuro2a presumably has no physiological relevance in vivo. Notably, the Mut APC2 proteins in Neuro2a were partially co-localized with the lysosome marker LAMP2 or the autophagy-related marker LC3 (Figures S4A and S4B). Using a series of deletion mutations, we identified an amino acid region (1821–1900, named region S) that was required for proper distribution along the microtubules as well as their stabilization (Figures S5A and S5B). The loss of function of ΔC hAPC2 was restored by the fusion of region S at the C terminus of ΔC in cultured cells (Figures S5C and S5D). Based on these results, we concluded that both Mut hAPC2 and ΔC hAPC2 lacking region S were loss-of-function mutants, and that the mutation in the APC2 gene was the probable cause of the clinical phenotypes of the patients. We determined whether the Apc2−/− mouse (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar) recapitulated the phenotypes of the patients. One characteristic feature of both patients (and Sotos syndrome patients) was a large head circumference (Cole and Hughes, 1994Cole T.R. Hughes H.E. Sotos syndrome: a study of the diagnostic criteria and natural history.J. Med. Genet. 1994; 31: 20-32Crossref PubMed Scopus (217) Google Scholar, Sotos et al., 1964Sotos J.F. Dodge P.R. Muirhead D. Crawford J.D. Talbot N.B. Cerebral gigantism in childhood. A syndrome of excessively rapid growth and acromegalic features and a nonprogressive neurologic disorder.N. Engl. J. Med. 1964; 271: 109-116Crossref PubMed Scopus (276) Google Scholar, Tatton-Brown et al., 2012Tatton-Brown K. Cole T.R.P. Rahman N. Sotos Syndrome. GeneReviews, 2012Google Scholar). Micro-computed tomography (micro-CT) imaging of live mice revealed that Apc2−/− mice had an abnormal head shape (Figure S6): the skull length was shortened without a change in the skull height (i.e., the ratio of the skull height to length was increased), whereas the ratio of the skull circumference to body length was increased. However, the body length was shortened, as described in our previous study (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). These CT appearances appeared to resemble the large head and head circumference of our patients. MRI studies have revealed that patients with Sotos syndrome often have dilated ventricles in the brain (Cecconi et al., 2005Cecconi M. Forzano F. Milani D. Cavani S. Baldo C. Selicorni A. Pantaleoni C. Silengo M. Ferrero G.B. Scarano G. et al.Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth.Am. J. Med. Genet. A. 2005; 134: 247-253Crossref PubMed Scopus (58) Google Scholar, Schaefer et al., 1997Schaefer G.B. Bodensteiner J.B. Buehler B.A. Lin A. Cole T.R. The neuroimaging findings in Sotos syndrome.Am. J. Med. Genet. 1997; 68: 462-465Crossref PubMed Scopus (107) Google Scholar). We observed prominent lateral ventricles in the brain of the male patient by MRI (Figure 4A). T2-weighted axial sections showed dilation in both lateral ventricles, which was particularly prominent in the right ventricle (Figure 4A, left). T1-weighted axial and T2-weighted coronal sections confirmed ventricular dilation with a prominent trigone region (Figure 4A, middle and right, respectively). These results further supported the diagnosis of Sotos syndrome in our patients. CT imaging of the heads of Apc2−/− mice stained with contrast material revealed that the brains also had dilated ventricles, similar to what was observed in our patients (and Sotos syndrome patients) (Figure 4B). The ventricles of Apc2−/− mice were significantly larger than those of WT and Apc2+/− mice (∼1.8-fold larger; Figure 4B), though marked variations in the size of the ventricles were observed among Apc2−/− mice. Agenesis or hypoplasia of the corpus callosum has occasionally been observed in Sotos syndrome patients (Cecconi et al., 2005Cecconi M. Forzano F. Milani D. Cavani S. Baldo C. Selicorni A. Pantaleoni C. Silengo M. Ferrero G.B. Scarano G. et al.Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth.Am. J. Med. Genet. A. 2005; 134: 247-253Crossref PubMed Scopus (58) Google Scholar, Schaefer et al., 1997Schaefer G.B. Bodensteiner J.B. Buehler B.A. Lin A. Cole T.R. The neuroimaging findings in Sotos syndrome.Am. J. Med. Genet. 1997; 68: 462-465Crossref PubMed Scopus (107) Google Scholar). The thickness of the corpus callosum was significantly reduced in Apc2−/− mice (∼75% that of WT mice; Figure 4C). However, our male patient did not have marked defects in the corpus callosum (data not shown). We conducted a series of behavioral tests on Apc2-deficient mice to determine whether they exhibited any intellectual or behavioral defects. In the open-field test, horizontal locomotion (number of crossings) in the total area was decreased by repeated exposure to the test environment in all genotypes (Figure 5A). However, the frequency with which Apc2+/− and Apc2−/− mice entered the central area was significantly lower than that observed for WT mice during the first open-field exposure (Figure 5B). No other significant differences were observed in the general behavior of Apc2-deficient mice, including exploratory rearing (Figure 5C). We then performed a novel-object exploration test using the same open field, to which the mice were already well habituated. The exploratory activity of Apc2−/− mice toward the novel object on day 1 was significantly higher than that of WT mice, and these high activity levels were maintained on day 2 (Figures 5D and 5E). Apc2−/− mice also exhibited enhanced exploratory activity in the elevated-plus maze test (Figure 5F). Since no genotypic difference was observed in the time spent in the open arms (Figure 5G), the increase in exploratory activity was not associated with a reduction in fear or anxiety toward the aversive environment. In a social-behavior test, Apc2−/− mice showed a significantly increased duration of interaction with the social target (an unfamiliar mouse), but also an increased interaction with an inanimate cage (Figure 5H). Therefore, no significant difference was observed in social versus inanimate preferences among genotypes (Figure 5I), which suggested that the deficiency in Apc2 did not affect social interactions, but rather solely increased spontaneous exploratory activity. Thus, the non-autistic behavioral abnormalities observed in Apc2-deficient mice also resembled the clinical features of our patients. We conducted a Morris water-maze analysis to evaluate spatial learning and memory abilities. All genotypes showed similar escape latencies in the visible-platform test (Figure 6A), which indicated that the motor (swim) and sensory (vision) functions of Apc2-deficient mice were normal. However, Apc2−/− mice clearly exhibited impaired learning abilities in the hidden-platform test and did not reach the criterion of learning in a total of 42 trials. Approximately 20 trials were sufficient for normal mice (Figure 6B). After the mice completed the hidden-platform task, we performed a probe test by removing the platform from the swimming pool. Apc2−/− mice showed a preference for the target quadrant, similarly to WT mice (Figure 6C), but performed a significantly lower number of crossings than WT mice (Figure 6D). Heatmap analyses of occupancy times clearly indicated that the Apc2−/− mice did not learn the platform location accurately (Figure 6E), and there were no genotypic differences in swim speed (Figure 6F). These results indicated that Apc2-deficient mice had impaired learning-memory function. Apc2−/− mice consistently showed impaired learning and memory abilities in the conditioned-fear test, in which mice learned to avoid entering a dark compartment in a light/dark avoidance apparatus after receiving one session of footshocks in the dark compartment (Figure 6G). Thus, we observed a high degree of concordance between our patients and these knockout mice with regard to brain dysfunction. NSD1 has been identified as the primary gene responsible for Sotos syndrome. It acts as a transcriptional regulator that modifies the expression of downstream genes that are more directly involved in the normal growth and development of several tissues, including the brain. We speculated that APC2, which is involved in brain development, may be a major downstream target of NSD1 in the nervous system. To test this hypothesis, we examined the effects of the knockdown of Nsd1 on the expression of Apc2 using primary mouse cortical cells. We tested two small hairpin RNA (shRNA) constructs directed toward different target sites in Nsd1, together with a control scramble shRNA construct. The expression level of Nsd1 was reduced by ∼40% (Figures 7A and 7B ). The knockdown of Nsd1 significantly reduced the expression of Apc2 mRNA to ∼40% of the control level (Figures 7A and 7B). Under the same experimental conditions, the expression of Apc mRNA, a member of the same gene family as Apc2, was not affected at all (Figures 7A and 7B). Western blot analyses confirmed that the expression of APC2 was suppressed by the knockdown of Nsd1: the knockdown of Nsd1 reduced the expression of the APC2 protein to ∼50% of the control level (Figure 7C). These results suggested that Apc2 is a downstream effector gene of Nsd1. We tested this possibility via in utero electroporation of miRNA (Baek et al., 2014Baek S.T. Kerjan G. Bielas S.L. Lee J.E. Fenstermaker A.G. Novarino G. Gleeson J.G. Off-target effect of doublecortin family shRNA on neuronal migration associated with endogenous microRNA dysregulation.Neuron. 2014; 82: 1255-1262Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) in order to examine the effects of Nsd1 knockdown on the migration of cortical neurons, because Apc2-deficient mice have marked defects in neuronal migration and layering in some brain regions, including the cerebral cortex (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). When control scrambled miRNA was introduced into cortical progenitor cells at embryonic day 15.5 (E15.5), most of the transfected neurons migrated normally to layer II-IV of the cortex at postnatal day 7 (P7) (Figure 7D). In contrast, a large population of neurons that were transfected with Apc2-miRNA remained in the lower layers (Figure 7D), which is in line with the phenotype observed in the Apc2−/− mouse brain (Shintani et al., 2012Shintani T. Takeuchi Y. Fujikawa A. Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.J. Neurosci. 2012; 32: 6468-6484Crossref PubMed Scopus (26) Google Scholar). Of note, cortical neurons transfected with Nsd1-miRNA showed a similar impairment in their migration (Figure 7D). In order to confirm that the migration defect induced by Nsd1 knockdown was attributed to the downregulated expression of Apc2, we performed rescue experiments. Importantly, co-electroporation of the Nsd1-miRNA construct with the Apc2-expression plasmid significantly rescued the migration defect in transfected neurons in the cortex (Figure 7E). This rescue effect was not observed with a control plasmid. Taken together, these results indicated that the expression of NSD1 controlled neuronal migration through the induction of APC2 expression. In the present study, we identified two siblings who were diagnosed with Sotos-like syndrome and presented with intellectual disability as well as characteristic facial features and a large head circumference. WES analyses revealed a homozygous single-nucleotide duplication in the APC2 gene (c.5199 dup) in these patients. This duplication resulted in a frameshift of 418 amino acids followed by a premature stop codon (p.[Lys1734Glnfs∗418]). Expression assays in cultured neuronal cells showed that the mutated APC2 protein in our patients was functionally null in its ability to regulate microtubule and actin dynamics. MRI and CT imaging demonstrated that Apc2-deficient mice and the male patient had an abnormal brain structure and head shape. In addition, a series of behavioral tests revealed that, like our patients, Apc2-deficient mice had impaired learning and memory abilities. Thus, the phenotypes observed in Apc2-deficient mice markedly resembled the clinical features present in the affected siblings, suggesting that a deficiency in APC2 caused the Sotos feature" @default.
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• W2054408230 date "2015-03-01" @default.
• W2054408230 modified "2023-09-23" @default.
• W2054408230 title "Loss-of-Function Mutation in APC2 Causes Sotos Syndrome Features" @default.
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