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Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis
Brain & Development xxx (2017) xxx–xxx www.elsevier.com/locate/braindev
Original article
Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis Hirofumi Kurata a,⇑,1, Kentaro Shirai a,b,1, Yoshiaki Saito a, Tetsuya Okazaki a,c, Koyo Ohno a, Masayoshi Oguri a,d, Kaori Adachi c, Eiji Nanba c, Yoshihiro Maegaki a a
Division of Child Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori University, Yonago, Japan b Department of Pediatrics, Tsuchiura Kyodo General Hospital, Tsuchiura, Japan c Division of Clinical Genetics, Tottori University Hospital, Yonago, Japan d Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, Yonago, Japan Received 11 April 2017; received in revised form 21 June 2017; accepted 10 July 2017
Abstract Purpose: To clarify the relationship between macrocephaly and neurodevelopmental disorders, as well as identify the prevalence of PTEN mutations in autism spectrum disorders with macrocephaly in Japan. Subjects and methods: Diagnostic and other medical information of children with macrocephaly younger than 4 years (n = 93) were collected for analysis. PTEN gene mutation analysis was conducted in another set of 16 macrocephalic individuals aged 3–22 years. Results: Sixteen macrocephalic children were associated with neurodevelopmental disorders, including autism spectrum disorders (ASDs) (n = 6), autistic traits (n = 5), intellectual disability (n = 5), attention deficit hyperactivity disorder (n = 1), developmental coordination disorders (n = 1), and language disorder (n = 1). Male gender was significantly linked to these disorders, whereas a family history and degree of macrocephaly were not significantly linked to the diagnosis. A novel mutation in the PTEN gene was identified in a 16-year-old girl with autism, mental retardation, language delay, extreme macrocephaly (+4.7 SD) with a prominent forehead, and digital minor anomalies. Conclusion: Children with macrocephaly, particularly males, are at a higher risk of neurodevelopmental disorders, rather than progressive etiologies, such as hydrocephalus and neurodegenerative disorders. The data provide a basis for routine health checks for young children in Japan, including the follow-up management and possible screening of PTEN mutations in children with ASDs and macrocephaly. Ó 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Autism spectrum disorders; Intellectual disability; Macrocephaly; Neurodevelopmental disorders; PTEN
1. Introduction ⇑ Corresponding author at: Division of Child Neurology, Depart-
ment of Brain and Neurosciences, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan. Fax: +81-85938-6779. E-mail address: [email protected] (H. Kurata). 1 These authors equally contributed to this article.
Macrocephaly in young children has been classically considered a risk factor for serious neurological diseases, including hydrocephalus accompanying brain tumors, leukodystrophies, and other neurodegenerative diseases such as GM2 gangliosidosis. Additionally, some reports revealed that macrocephaly is a biomarker of the
http://dx.doi.org/10.1016/j.braindev.2017.07.005 0387-7604/Ó 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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H. Kurata et al. / Brain & Development xxx (2017) xxx–xxx
autism spectrum disorders (ASDs), reported in 12–31% of autistic children [1–4]. In turn, approximately 3% of boys with macrocephaly were associated with ASDs [5]. The proportion of ASDs in children with macrocephaly, irrespective of their sex, or in populations outside England, has not been reported. Multiple genetic factors have been identified to cause ASDs [6–8]. Among others, mutations in the PTEN (phosphatase and tensin homolog) gene have been identified in individuals with autism accompanied by macrocephaly [9–11], which account for 1%–17% of ASDs with macrocephaly [9,10]. The protein encoded by PTEN gene was initially recognized as a tumor suppressor, which antagonizes the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and suppresses the proliferation of cancer cells through inhibition of the mammalian target of rapamycin (mTOR) pathway downstream of AKT [12]. Tuberous sclerosis complex (TSC) is caused by the activation of the mTOR pathway due to TSC1/2 mutations, and up to 50% of children with TSC manifest with ASDs [13]. mTOR inhibitors, such as rapamycin, can recover the impaired social interaction in TSC2+/ mice with simultaneous reduction of elevated phospho-S6K levels in the brain [14]. Furthermore, they can ameliorate autistic symptoms in adolescents with TSC [15] as well as abnormal behaviors in Pten knockout mice [16]. Thus, these agents may be potentially effective for the treatment of autistic children with macrocephaly due to PTEN mutations. With this background, our first aim was to confirm the correlation between macrocephaly and ASDs and their proportions in Japanese children to assist in diagnosis during health visits and to provide information for medical follow-up based on early intervention. Our second aim in this study was to determine the prevalence of children with macrocephaly associated with PTEN mutations in Japan to develop increased understanding for preparing a screening plan for the future. 2. Subjects and methods 2.1. Prevalence of neurodevelopmental disorders in macrocephalic children This study enrolled 93 Japanese children with macrocephaly, aged 1–44 months, who were referred to the Division of Child Neurology, Tottori University Hospital between January 2006 and August 2015. Two subjects already diagnosed with congenital hydrocephalus and megalencephalic leukoencephalopathy with subcortical cysts, confirmed before the initial referral, were excluded before enrollment. We defined macrocephaly as an occipito-frontal head circumference (HC) above 2 standard deviations (SD) from the mean for age. Each subject was examined by experienced child neurologists
to diagnosis developmental disorders. Diagnoses were initially based on DSM-IV criteria and were converted to the classification in DSM-V for description in this study, with diagnoses of pervasive developmental disorder and mental retardation converted to ASDs and intellectual disability (ID), respectively. An initial diagnosis of Asperger syndrome was converted to ASDs in several cases. Subjects who were suspected to have ASDs and had the potential for social impairment, but definite diagnosis was withheld due to young age as well as those who did not completely meet the contents of criterion A in the DSM-V diagnostic criteria for ASDs were classified as having ‘‘autistic traits.” ID was defined by an intelligence quotient (IQ) score or developmental quotient (DQ) below 70. IQ/DQ was determined based on the results of the Wechsler Intelligence Scale for Children-Fourth Edition, revised version of the Kyoto Scale of Psychological Development, Kinder infant development scale, or Enjoji developmental scale. Information on the family history of macrocephaly, accompanying conditions, and the imaging findings of cranial computed tomography (CT) or the brain magnetic resonance imaging (MRI) were also collected. The study design was approved by the ethical committee of the Tottori University Faculty of Medicine. 2.2. PTEN mutation analysis The macrocephalic subjects with ASDs and/or ID who were referred to our department between 2004 and 2012 (16 subjects with ASDs (n = 15)/autistic traits (n = 1)) were enrolled into this study after having written informed consent from their parents. Three subjects with ASDs/autistic traits in the prevalence study were also included in this study. For the genetic diagnosis, blood samples were obtained from the patients, parents, and siblings after written informed consent was obtained from the parents. Genomic DNA was extracted from the blood samples using a standard protocol. All nine exons and exon–intron junctions of the PTEN gene were amplified from genomic DNA by PCR using primers designed with primer software (Genetyx software, Genetyx, Shibuya, Japan). The primers were designed to include an intron of 50 bp surrounding the exon. The sequencing was performed using forward and reverse primers. All the primer sequences are available on request. Sequencing was performed with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3500 L Genetic Analyzer capillary sequencer (Life Technologies, Carlsbad, CA, USA). Publicly available prediction programs, such as PolyPhen-2 and SIFT algorithm, were used to analyze the detected variants [17,18]. The study design was approved by the ethical committee of the Tottori University Faculty of Medicine.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
H. Kurata et al. / Brain & Development xxx (2017) xxx–xxx
2.3. Statistical analysis For statistical analysis, a chi-square and Fisher’s exact test were used to assess group differences in nonnumeric issues, and a Mann-Whitney U test was used to compare continuous variables. Multivariate logistic regression analysis was performed to identify the predictive factors for macrocephalic male and female. Statistical analyses were performed using SPSS Statistics 24.0 (IBM, Tokyo, Japan). All statistical tests were twotailed, and p value <0.05 was considered to indicate statistical significance. 3. Results 3.1. Prevalence of neurodevelopmental disorders in macrocephalic children Ninety-three children were referred from routine health checks or other hospitals and departments for assessment by child neurologists (Table S1). Retrospective data of 93 macrocephalic patients (61 males and 32 females, ratio 1.9:1) were collected (Table 1). A total of 41 patients had a familial history of macrocephaly and 16 patients (14 males and 2 females) were diagnosed with certain types of neurodevelopmental disorders, overlapping with ASDs (n = 6), autistic traits (n = 5), intellectual disability (n = 5), attention deficit hyperactivity disorder (n = 1), developmental coordination disorders (n = 1), and language disorder (n = 1). In this study, the proportion of neurodevelopmental disorders was 17% (16/93) in children with macrocephaly and
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23% (14/61) in males with macrocephaly. Specifically, ASDs (10% in males) or its traits (8% in males) accounted for two-thirds of these morbidities. Twelve of the patients with macrocephaly had accompanying conditions, including facial, digital, cardiac or pulmonary anomalies, benign infantile convulsion, and hypothyroidism (Table S2). A girl with ASDs and ID was diagnosed with Sotos syndrome by identification of a NSD1 gene mutation after referral to our institution. Another girl was clinically diagnosed as having megalocornea-mental retardation syndrome. To evaluate the etiology of the macrocephalic patients, cranial CT and MRI were first performed in 76 and 7 patients, respectively. Cranial CT revealed left-sided chronic subdural hematoma in one male patient, who was referred from a 3 months old health check. He was asymptomatic and treated by careful monitoring and observation. In the other 75 patients, ventriculomegaly (n = 13), suspected arachnoid cyst (n = 8), and enlargement of the subarachnoid spaces or craniocerebral disproportion (n = 10) were observed. MRI for an additional 15 patients was examined for follow-up imaging after the cranial CT to avoid repeated radiation exposure from the CT scans. MRI revealed the chronic subdural hematoma in one patient as mentioned above. In other 21 patients, ventriculomegaly (n = 7), enlargement of the subarachnoid spaces or craniocerebral disproportion (n = 3), arachnoid cyst (n = 2), pineal cyst (n = 1), thin corpus callosum (n = 1), and small cyst near the foramen of Monro (n = 1) were observed. No cases required immediate intervention after performing MRI or CT.
Table 1 Demographic and clinical characteristics of macrocephalic children. Parameter
Macrocephalic patients (n = 93)
Age (months) Gender (male/female) Familial macrocephaly (male/female) Mean HC at first visit (SD) Diagnosis Macrocephalic subjects without neurodevelopmental disorders (male/female) Neurodevelopmental Disorders (male/female) ID (male/female)
17.7 ± 10.9* (1–44 months) 61/32 41 (22/19) 2.5 ± 0.64*
ADHD (male/female) DCD (male/female) ASD (male/female) Autistic traits (male/female) LD (male/female) Number of cranial CT (male/female) Number of brain MRI (male/female) Patients with underlying complications, anomalies (male/female)
1 (1/0) 1 (0/1) 6 (6/0) 5 (5/0) 1 (1/0) 76 (50/26) 22 (16/6) 12 (4/8)
77 (47/30) 16 (14/2) 5 (4/1)
Overlapping with autistic traits (n = 1), ASD (n = 1) Overlapping with autistic triats (n = 1) Overlapping with ID (n = 1) Overlapping with ID (n = 1), ADHD (n = 1)
Abbreviations: ID, intellectutual disability; ASD, Autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; LD, Language disorder; DCD, developmental coordination disorders; SD, standard deviation; HC, head circumference. * Data are presented as mean ± SD.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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Table 2 Univariate analysis that examined the influence of the gender.
Age (months) Familial macrocephaly Diagnosis Neurodevelopmental Disorders ID ASD ASD or Autistic traits Patients with accompanying conditions HC at first visit (SD) Number of cranial CT
Macrocephalic male (n = 61)
Macrocephalic female (n = 32)
P-Value
18.6 22
16 19
0.656* 0.031
14
2
0.043
4
1
6 11
0 0
4
8
0.02
2.5 49
2.5 27
0.637* 0.631
Overlapping with autistic traits (n = 1), ASD (n = 1) Overlapping with ID (n = 1) Overlapping with ID (n = 2), ADHD (n = 1)
0.657 0.09 0.014
Abbreviations: HC, head circumference; ID, intellectutual disability; ASD, Autism spectrum disorder; SD, standard deviation. * Data are expressed as the mean and analyzed using the Mann Whitney U test. The others were analyzed by the chi-squre and fisher’s exact test.
Table 3 Multivariate logistic regression analysis that examined the influence of gender. Possible predictor
Odds ratio
95% confidence interval
P-Value
Familial macrocephaly Patients with accompanying conditions Diagnosis of neurodevelopmental Disorders
3.029 8.49 0.162
1.152–7.969 1.877–38.389 0.028–0.949
0.025 0.005 0.044
Univariate analysis indicated that neurodevelopmental disorders, ASDs, and autistic traits were significantly linked to the male gender. Familial macrocephaly and accompanying complications, including congenital anomalies, were significantly linked to the female gender (Table 2). Of the 41 participants with familial macrocephaly, five had neurodevelopmental disorders, including ASDs (n = 3), autistic traits (n = 1), and intellectual disabilities (n = 2, overlapping with autistic traits [n = 1]). The degree of macrocephaly, a family history of macrocephaly, or the presence of accompanying conditions did not reach a significant link to other variables, including neurodevelopmental disorders (not shown). Neurodevelopmental disorders were rare in subjects with findings of ventriculomegaly, an enlargement of subarachnoid space and others (Table S4). Multivariate analysis revealed that the following three factors were independently associated with gender in macrocephalic children: neurodevelopmental disorders were significantly more common in males, familial history of macrocephaly and accompanying conditions were more common in females (Table 3).
(p.M1V), in a 16-year-old girl (Fig. S1). The mutation was predicted to be pathogenic based upon the in silico programs SIFT: (damaging, score 0.004), and Polyphen2: with a possible damaging score: 0.713 (sensitivity: 0.86, specificity: 0.92). One mutation among 16 subjects represented 6% with macrocephaly and ASDs. The patient with this mutation presented with ASDs, intellectual disability, language delay, extreme macrocephaly (+4.1 SD) with a prominent forehead, and digital minor anomalies (hypoplasia of IVth and Vth fingers). A cranial CT at 11 years of age was unremarkable. Although her father and younger brother had the same PTEN mutation, macrocephaly (head circumference: father, 65.5 cm; brother, 58 cm [+2.6 SD] at the age of 12 years), and the digital minor anomalies identical to the patient, they appeared intellectually normal. The brother had joined a class with usual educational level at a junior high school. They did not agree with the assessment of their autistic traits. There was no family history that was suggestive of an underlying PTEN hamartoma tumor syndrome. 4. Discussion
3.2. PTEN mutation analysis Among the 16 individuals (15 ASDs with/without ID, one case with autistic traits), head circumference ranged from 2.0 to 5.2 SD above the mean (Table S3). We identified a novel PTEN missense mutation, c.1A>G
This is the first report of the proportion of neurodevelopmental disorders, including ASDs, in Japanese children, which should be recognized in routine health checks of young children. A potential overestimation is undeniable due to the bias in referral of subjects with
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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severe manifestations to the university hospital. A similar study in England, with enrollment of all macrocephalic children at a routine health check, identified the diagnosis of ASDs in 3% (9/306) of males in the general population and 5.7% (9/158) of children referred to a pediatrician or pediatric psychiatrist by the routine health check [5]. Our data regarding the identification of ASDs in 9.8% (6/61) of males with macrocephaly referred to the university hospital was comparable to that of the latter proportion in the abovementioned study; a population-based study should also be completed in Japan. Male predominance has been well established in ASDs with/without macrocephaly. The significance of ID without ASDs in macrocephalic children was also confirmed in this study. Familial history of macrocephaly has been regarded as a hallmark of ‘‘benign” condition [19,20], but it did not warrant normal socialintellectual development in many cases in this study. In fact, either of the parents had macrocephaly in 33% of autistic children [21]. A learning disability may be common in familial macrocephaly [22], although this could not be assessed in the present study due to the lack of subjects with a learning disability. Notably, subjects with ASDs or autistic traits did not have abnormalities on brain neuroimaging. Radiation exposure in cranial CT examinations in children has a risk of development of malignancy. The calculated risk from one cranial CT in children younger than 10 years is assumed 1/1000 for leukemia and brain tumors [23]. MRI should first be performed for children with macrocephaly, and CT scan should be limited to children with neurological abnormalities other than ASDs/ID. In previous studies, the prevalence of the PTEN gene mutation in ASDs subjects was 5 out of 60 (8.3%) [11]. The prevalence in macrocephalic ASDs subjects ranged from 3 out of 18 (17%) [9] to one out of 88 (1%) [10]. A recent report from Japan identified 3 subjects with PTEN mutations out of 13 (23%) with macrocephaly and developmental delay or epilepsy [24], which is higher than our results of one mutation in 16 subjects (6%) with macrocephaly and ASDs. These varying results suggest the need for further hallmarks of PTEN-related ASDs with macrocephaly. The degree of macrocephaly in ASDs patients due to PTEN mutations is often >+4.0 SD [9], common with the patients in this study. However, a head circumference as low as +2.9 SD was also reported [11]. Male predominance was not noted in the PTEN-related disorders [24], as it was seen in the female patient in the present study. The significance of minor congenital anomalies, including facial dysmorphism, is also uncertain, as polydactyly and other digital abnormalities have been previously described in some patients in PTEN-related syndromes [10,24]. Screening for an elevated phosphorylated S6 ribosomal protein, which represents the activation of
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the PTEN/mTOR pathway, in lymphoblastoid cells [24] may be a promising alternative to identify the PTEN-related ASDs and macrocephaly. The mutation c.1A>G (p.M1V) was assumed to result in the loss of the start codon of the transcript. While we do not have the functional data on the effects of this mutation on the protein, the mutation was predicted to be pathogenic based upon the in silico programs. In addition, two patients with ASDs and macrocephaly had a missense mutation of PTEN in the start codon, c.3G>T (p.M1I), which functionally resulted in the upregulation of the AKT and MAPK pathways confirmed by increased P-AKT and PMAPK42/44 in blood samples [25]. The same pathogenic mutations of c.1A>G have been identified in several genetic disorders including, Nager syndrome, hypotrichosis simplex [26], Leigh syndrome, cranioectodermal dysplasia 3, and Sensenbrenner syndrome [27]. These support that c.1A>G is pathognomonic. The lack of intellectual disability or epilepsy in the father and brother may suggest certain epigenetic factors for the phenotype of PTEN mutations. Indeed, similar asymptomatic fathers with PTEN mutations identical to their ASDs sons have been reported [11]. Children with macrocephaly, particularly males, are at a higher risk of neurodevelopmental disorders (14/61, 22%), including ASDs, (6/61, 9.8%) at the referral from routine health checks. A family history of macrocephaly does not always warrant normal socialintellectual development. We identified one female with PTEN-related ASDs (6.2%). Hallmarks of PTENrelated ASDs have not been established, including its association with the male sex. Our data provide a basis for routine health checks for young children with macrocephaly in Japan and suggest the need for further population-based studies as well as the development of an effective screening plan for PTEN mutations in children with ASDs and macrocephaly. Conflict of interest All the authors claim no conflicts of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.braindev.2017.07.005. References [1] Sacco R, Gabriele S, Persico AM. Head circumference and brain size in autism spectrum disorder: a systematic review and metaanalysis. Psychiatry Res 2015;234:239–51. [2] O’Reilly H, Thie´baut FI, White SJ. Is macrocephaly a neural marker of a local bias in autism? Dev Cogn Neurosci 2013;6:149–54.
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Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
Original article
Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis Hirofumi Kurata a,⇑,1, Kentaro Shirai a,b,1, Yoshiaki Saito a, Tetsuya Okazaki a,c, Koyo Ohno a, Masayoshi Oguri a,d, Kaori Adachi c, Eiji Nanba c, Yoshihiro Maegaki a a
Division of Child Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori University, Yonago, Japan b Department of Pediatrics, Tsuchiura Kyodo General Hospital, Tsuchiura, Japan c Division of Clinical Genetics, Tottori University Hospital, Yonago, Japan d Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, Yonago, Japan Received 11 April 2017; received in revised form 21 June 2017; accepted 10 July 2017
Abstract Purpose: To clarify the relationship between macrocephaly and neurodevelopmental disorders, as well as identify the prevalence of PTEN mutations in autism spectrum disorders with macrocephaly in Japan. Subjects and methods: Diagnostic and other medical information of children with macrocephaly younger than 4 years (n = 93) were collected for analysis. PTEN gene mutation analysis was conducted in another set of 16 macrocephalic individuals aged 3–22 years. Results: Sixteen macrocephalic children were associated with neurodevelopmental disorders, including autism spectrum disorders (ASDs) (n = 6), autistic traits (n = 5), intellectual disability (n = 5), attention deficit hyperactivity disorder (n = 1), developmental coordination disorders (n = 1), and language disorder (n = 1). Male gender was significantly linked to these disorders, whereas a family history and degree of macrocephaly were not significantly linked to the diagnosis. A novel mutation in the PTEN gene was identified in a 16-year-old girl with autism, mental retardation, language delay, extreme macrocephaly (+4.7 SD) with a prominent forehead, and digital minor anomalies. Conclusion: Children with macrocephaly, particularly males, are at a higher risk of neurodevelopmental disorders, rather than progressive etiologies, such as hydrocephalus and neurodegenerative disorders. The data provide a basis for routine health checks for young children in Japan, including the follow-up management and possible screening of PTEN mutations in children with ASDs and macrocephaly. Ó 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Autism spectrum disorders; Intellectual disability; Macrocephaly; Neurodevelopmental disorders; PTEN
1. Introduction ⇑ Corresponding author at: Division of Child Neurology, Depart-
ment of Brain and Neurosciences, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan. Fax: +81-85938-6779. E-mail address: [email protected] (H. Kurata). 1 These authors equally contributed to this article.
Macrocephaly in young children has been classically considered a risk factor for serious neurological diseases, including hydrocephalus accompanying brain tumors, leukodystrophies, and other neurodegenerative diseases such as GM2 gangliosidosis. Additionally, some reports revealed that macrocephaly is a biomarker of the
http://dx.doi.org/10.1016/j.braindev.2017.07.005 0387-7604/Ó 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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autism spectrum disorders (ASDs), reported in 12–31% of autistic children [1–4]. In turn, approximately 3% of boys with macrocephaly were associated with ASDs [5]. The proportion of ASDs in children with macrocephaly, irrespective of their sex, or in populations outside England, has not been reported. Multiple genetic factors have been identified to cause ASDs [6–8]. Among others, mutations in the PTEN (phosphatase and tensin homolog) gene have been identified in individuals with autism accompanied by macrocephaly [9–11], which account for 1%–17% of ASDs with macrocephaly [9,10]. The protein encoded by PTEN gene was initially recognized as a tumor suppressor, which antagonizes the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and suppresses the proliferation of cancer cells through inhibition of the mammalian target of rapamycin (mTOR) pathway downstream of AKT [12]. Tuberous sclerosis complex (TSC) is caused by the activation of the mTOR pathway due to TSC1/2 mutations, and up to 50% of children with TSC manifest with ASDs [13]. mTOR inhibitors, such as rapamycin, can recover the impaired social interaction in TSC2+/ mice with simultaneous reduction of elevated phospho-S6K levels in the brain [14]. Furthermore, they can ameliorate autistic symptoms in adolescents with TSC [15] as well as abnormal behaviors in Pten knockout mice [16]. Thus, these agents may be potentially effective for the treatment of autistic children with macrocephaly due to PTEN mutations. With this background, our first aim was to confirm the correlation between macrocephaly and ASDs and their proportions in Japanese children to assist in diagnosis during health visits and to provide information for medical follow-up based on early intervention. Our second aim in this study was to determine the prevalence of children with macrocephaly associated with PTEN mutations in Japan to develop increased understanding for preparing a screening plan for the future. 2. Subjects and methods 2.1. Prevalence of neurodevelopmental disorders in macrocephalic children This study enrolled 93 Japanese children with macrocephaly, aged 1–44 months, who were referred to the Division of Child Neurology, Tottori University Hospital between January 2006 and August 2015. Two subjects already diagnosed with congenital hydrocephalus and megalencephalic leukoencephalopathy with subcortical cysts, confirmed before the initial referral, were excluded before enrollment. We defined macrocephaly as an occipito-frontal head circumference (HC) above 2 standard deviations (SD) from the mean for age. Each subject was examined by experienced child neurologists
to diagnosis developmental disorders. Diagnoses were initially based on DSM-IV criteria and were converted to the classification in DSM-V for description in this study, with diagnoses of pervasive developmental disorder and mental retardation converted to ASDs and intellectual disability (ID), respectively. An initial diagnosis of Asperger syndrome was converted to ASDs in several cases. Subjects who were suspected to have ASDs and had the potential for social impairment, but definite diagnosis was withheld due to young age as well as those who did not completely meet the contents of criterion A in the DSM-V diagnostic criteria for ASDs were classified as having ‘‘autistic traits.” ID was defined by an intelligence quotient (IQ) score or developmental quotient (DQ) below 70. IQ/DQ was determined based on the results of the Wechsler Intelligence Scale for Children-Fourth Edition, revised version of the Kyoto Scale of Psychological Development, Kinder infant development scale, or Enjoji developmental scale. Information on the family history of macrocephaly, accompanying conditions, and the imaging findings of cranial computed tomography (CT) or the brain magnetic resonance imaging (MRI) were also collected. The study design was approved by the ethical committee of the Tottori University Faculty of Medicine. 2.2. PTEN mutation analysis The macrocephalic subjects with ASDs and/or ID who were referred to our department between 2004 and 2012 (16 subjects with ASDs (n = 15)/autistic traits (n = 1)) were enrolled into this study after having written informed consent from their parents. Three subjects with ASDs/autistic traits in the prevalence study were also included in this study. For the genetic diagnosis, blood samples were obtained from the patients, parents, and siblings after written informed consent was obtained from the parents. Genomic DNA was extracted from the blood samples using a standard protocol. All nine exons and exon–intron junctions of the PTEN gene were amplified from genomic DNA by PCR using primers designed with primer software (Genetyx software, Genetyx, Shibuya, Japan). The primers were designed to include an intron of 50 bp surrounding the exon. The sequencing was performed using forward and reverse primers. All the primer sequences are available on request. Sequencing was performed with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3500 L Genetic Analyzer capillary sequencer (Life Technologies, Carlsbad, CA, USA). Publicly available prediction programs, such as PolyPhen-2 and SIFT algorithm, were used to analyze the detected variants [17,18]. The study design was approved by the ethical committee of the Tottori University Faculty of Medicine.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
H. Kurata et al. / Brain & Development xxx (2017) xxx–xxx
2.3. Statistical analysis For statistical analysis, a chi-square and Fisher’s exact test were used to assess group differences in nonnumeric issues, and a Mann-Whitney U test was used to compare continuous variables. Multivariate logistic regression analysis was performed to identify the predictive factors for macrocephalic male and female. Statistical analyses were performed using SPSS Statistics 24.0 (IBM, Tokyo, Japan). All statistical tests were twotailed, and p value <0.05 was considered to indicate statistical significance. 3. Results 3.1. Prevalence of neurodevelopmental disorders in macrocephalic children Ninety-three children were referred from routine health checks or other hospitals and departments for assessment by child neurologists (Table S1). Retrospective data of 93 macrocephalic patients (61 males and 32 females, ratio 1.9:1) were collected (Table 1). A total of 41 patients had a familial history of macrocephaly and 16 patients (14 males and 2 females) were diagnosed with certain types of neurodevelopmental disorders, overlapping with ASDs (n = 6), autistic traits (n = 5), intellectual disability (n = 5), attention deficit hyperactivity disorder (n = 1), developmental coordination disorders (n = 1), and language disorder (n = 1). In this study, the proportion of neurodevelopmental disorders was 17% (16/93) in children with macrocephaly and
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23% (14/61) in males with macrocephaly. Specifically, ASDs (10% in males) or its traits (8% in males) accounted for two-thirds of these morbidities. Twelve of the patients with macrocephaly had accompanying conditions, including facial, digital, cardiac or pulmonary anomalies, benign infantile convulsion, and hypothyroidism (Table S2). A girl with ASDs and ID was diagnosed with Sotos syndrome by identification of a NSD1 gene mutation after referral to our institution. Another girl was clinically diagnosed as having megalocornea-mental retardation syndrome. To evaluate the etiology of the macrocephalic patients, cranial CT and MRI were first performed in 76 and 7 patients, respectively. Cranial CT revealed left-sided chronic subdural hematoma in one male patient, who was referred from a 3 months old health check. He was asymptomatic and treated by careful monitoring and observation. In the other 75 patients, ventriculomegaly (n = 13), suspected arachnoid cyst (n = 8), and enlargement of the subarachnoid spaces or craniocerebral disproportion (n = 10) were observed. MRI for an additional 15 patients was examined for follow-up imaging after the cranial CT to avoid repeated radiation exposure from the CT scans. MRI revealed the chronic subdural hematoma in one patient as mentioned above. In other 21 patients, ventriculomegaly (n = 7), enlargement of the subarachnoid spaces or craniocerebral disproportion (n = 3), arachnoid cyst (n = 2), pineal cyst (n = 1), thin corpus callosum (n = 1), and small cyst near the foramen of Monro (n = 1) were observed. No cases required immediate intervention after performing MRI or CT.
Table 1 Demographic and clinical characteristics of macrocephalic children. Parameter
Macrocephalic patients (n = 93)
Age (months) Gender (male/female) Familial macrocephaly (male/female) Mean HC at first visit (SD) Diagnosis Macrocephalic subjects without neurodevelopmental disorders (male/female) Neurodevelopmental Disorders (male/female) ID (male/female)
17.7 ± 10.9* (1–44 months) 61/32 41 (22/19) 2.5 ± 0.64*
ADHD (male/female) DCD (male/female) ASD (male/female) Autistic traits (male/female) LD (male/female) Number of cranial CT (male/female) Number of brain MRI (male/female) Patients with underlying complications, anomalies (male/female)
1 (1/0) 1 (0/1) 6 (6/0) 5 (5/0) 1 (1/0) 76 (50/26) 22 (16/6) 12 (4/8)
77 (47/30) 16 (14/2) 5 (4/1)
Overlapping with autistic traits (n = 1), ASD (n = 1) Overlapping with autistic triats (n = 1) Overlapping with ID (n = 1) Overlapping with ID (n = 1), ADHD (n = 1)
Abbreviations: ID, intellectutual disability; ASD, Autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; LD, Language disorder; DCD, developmental coordination disorders; SD, standard deviation; HC, head circumference. * Data are presented as mean ± SD.
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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Table 2 Univariate analysis that examined the influence of the gender.
Age (months) Familial macrocephaly Diagnosis Neurodevelopmental Disorders ID ASD ASD or Autistic traits Patients with accompanying conditions HC at first visit (SD) Number of cranial CT
Macrocephalic male (n = 61)
Macrocephalic female (n = 32)
P-Value
18.6 22
16 19
0.656* 0.031
14
2
0.043
4
1
6 11
0 0
4
8
0.02
2.5 49
2.5 27
0.637* 0.631
Overlapping with autistic traits (n = 1), ASD (n = 1) Overlapping with ID (n = 1) Overlapping with ID (n = 2), ADHD (n = 1)
0.657 0.09 0.014
Abbreviations: HC, head circumference; ID, intellectutual disability; ASD, Autism spectrum disorder; SD, standard deviation. * Data are expressed as the mean and analyzed using the Mann Whitney U test. The others were analyzed by the chi-squre and fisher’s exact test.
Table 3 Multivariate logistic regression analysis that examined the influence of gender. Possible predictor
Odds ratio
95% confidence interval
P-Value
Familial macrocephaly Patients with accompanying conditions Diagnosis of neurodevelopmental Disorders
3.029 8.49 0.162
1.152–7.969 1.877–38.389 0.028–0.949
0.025 0.005 0.044
Univariate analysis indicated that neurodevelopmental disorders, ASDs, and autistic traits were significantly linked to the male gender. Familial macrocephaly and accompanying complications, including congenital anomalies, were significantly linked to the female gender (Table 2). Of the 41 participants with familial macrocephaly, five had neurodevelopmental disorders, including ASDs (n = 3), autistic traits (n = 1), and intellectual disabilities (n = 2, overlapping with autistic traits [n = 1]). The degree of macrocephaly, a family history of macrocephaly, or the presence of accompanying conditions did not reach a significant link to other variables, including neurodevelopmental disorders (not shown). Neurodevelopmental disorders were rare in subjects with findings of ventriculomegaly, an enlargement of subarachnoid space and others (Table S4). Multivariate analysis revealed that the following three factors were independently associated with gender in macrocephalic children: neurodevelopmental disorders were significantly more common in males, familial history of macrocephaly and accompanying conditions were more common in females (Table 3).
(p.M1V), in a 16-year-old girl (Fig. S1). The mutation was predicted to be pathogenic based upon the in silico programs SIFT: (damaging, score 0.004), and Polyphen2: with a possible damaging score: 0.713 (sensitivity: 0.86, specificity: 0.92). One mutation among 16 subjects represented 6% with macrocephaly and ASDs. The patient with this mutation presented with ASDs, intellectual disability, language delay, extreme macrocephaly (+4.1 SD) with a prominent forehead, and digital minor anomalies (hypoplasia of IVth and Vth fingers). A cranial CT at 11 years of age was unremarkable. Although her father and younger brother had the same PTEN mutation, macrocephaly (head circumference: father, 65.5 cm; brother, 58 cm [+2.6 SD] at the age of 12 years), and the digital minor anomalies identical to the patient, they appeared intellectually normal. The brother had joined a class with usual educational level at a junior high school. They did not agree with the assessment of their autistic traits. There was no family history that was suggestive of an underlying PTEN hamartoma tumor syndrome. 4. Discussion
3.2. PTEN mutation analysis Among the 16 individuals (15 ASDs with/without ID, one case with autistic traits), head circumference ranged from 2.0 to 5.2 SD above the mean (Table S3). We identified a novel PTEN missense mutation, c.1A>G
This is the first report of the proportion of neurodevelopmental disorders, including ASDs, in Japanese children, which should be recognized in routine health checks of young children. A potential overestimation is undeniable due to the bias in referral of subjects with
Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005
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severe manifestations to the university hospital. A similar study in England, with enrollment of all macrocephalic children at a routine health check, identified the diagnosis of ASDs in 3% (9/306) of males in the general population and 5.7% (9/158) of children referred to a pediatrician or pediatric psychiatrist by the routine health check [5]. Our data regarding the identification of ASDs in 9.8% (6/61) of males with macrocephaly referred to the university hospital was comparable to that of the latter proportion in the abovementioned study; a population-based study should also be completed in Japan. Male predominance has been well established in ASDs with/without macrocephaly. The significance of ID without ASDs in macrocephalic children was also confirmed in this study. Familial history of macrocephaly has been regarded as a hallmark of ‘‘benign” condition [19,20], but it did not warrant normal socialintellectual development in many cases in this study. In fact, either of the parents had macrocephaly in 33% of autistic children [21]. A learning disability may be common in familial macrocephaly [22], although this could not be assessed in the present study due to the lack of subjects with a learning disability. Notably, subjects with ASDs or autistic traits did not have abnormalities on brain neuroimaging. Radiation exposure in cranial CT examinations in children has a risk of development of malignancy. The calculated risk from one cranial CT in children younger than 10 years is assumed 1/1000 for leukemia and brain tumors [23]. MRI should first be performed for children with macrocephaly, and CT scan should be limited to children with neurological abnormalities other than ASDs/ID. In previous studies, the prevalence of the PTEN gene mutation in ASDs subjects was 5 out of 60 (8.3%) [11]. The prevalence in macrocephalic ASDs subjects ranged from 3 out of 18 (17%) [9] to one out of 88 (1%) [10]. A recent report from Japan identified 3 subjects with PTEN mutations out of 13 (23%) with macrocephaly and developmental delay or epilepsy [24], which is higher than our results of one mutation in 16 subjects (6%) with macrocephaly and ASDs. These varying results suggest the need for further hallmarks of PTEN-related ASDs with macrocephaly. The degree of macrocephaly in ASDs patients due to PTEN mutations is often >+4.0 SD [9], common with the patients in this study. However, a head circumference as low as +2.9 SD was also reported [11]. Male predominance was not noted in the PTEN-related disorders [24], as it was seen in the female patient in the present study. The significance of minor congenital anomalies, including facial dysmorphism, is also uncertain, as polydactyly and other digital abnormalities have been previously described in some patients in PTEN-related syndromes [10,24]. Screening for an elevated phosphorylated S6 ribosomal protein, which represents the activation of
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the PTEN/mTOR pathway, in lymphoblastoid cells [24] may be a promising alternative to identify the PTEN-related ASDs and macrocephaly. The mutation c.1A>G (p.M1V) was assumed to result in the loss of the start codon of the transcript. While we do not have the functional data on the effects of this mutation on the protein, the mutation was predicted to be pathogenic based upon the in silico programs. In addition, two patients with ASDs and macrocephaly had a missense mutation of PTEN in the start codon, c.3G>T (p.M1I), which functionally resulted in the upregulation of the AKT and MAPK pathways confirmed by increased P-AKT and PMAPK42/44 in blood samples [25]. The same pathogenic mutations of c.1A>G have been identified in several genetic disorders including, Nager syndrome, hypotrichosis simplex [26], Leigh syndrome, cranioectodermal dysplasia 3, and Sensenbrenner syndrome [27]. These support that c.1A>G is pathognomonic. The lack of intellectual disability or epilepsy in the father and brother may suggest certain epigenetic factors for the phenotype of PTEN mutations. Indeed, similar asymptomatic fathers with PTEN mutations identical to their ASDs sons have been reported [11]. Children with macrocephaly, particularly males, are at a higher risk of neurodevelopmental disorders (14/61, 22%), including ASDs, (6/61, 9.8%) at the referral from routine health checks. A family history of macrocephaly does not always warrant normal socialintellectual development. We identified one female with PTEN-related ASDs (6.2%). Hallmarks of PTENrelated ASDs have not been established, including its association with the male sex. Our data provide a basis for routine health checks for young children with macrocephaly in Japan and suggest the need for further population-based studies as well as the development of an effective screening plan for PTEN mutations in children with ASDs and macrocephaly. Conflict of interest All the authors claim no conflicts of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.braindev.2017.07.005. References [1] Sacco R, Gabriele S, Persico AM. Head circumference and brain size in autism spectrum disorder: a systematic review and metaanalysis. Psychiatry Res 2015;234:239–51. [2] O’Reilly H, Thie´baut FI, White SJ. Is macrocephaly a neural marker of a local bias in autism? Dev Cogn Neurosci 2013;6:149–54.
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Please cite this article in press as: Kurata H et al. Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev (2017), http://dx.doi.org/10.1016/j.braindev.2017.07.005