Autism spectrum disorders (ASD) are childhood-onset neurodevelopmental disorders characterized by verbal communication impairments, social reciprocity deficits, and the presence of restricted interests and stereotyped behaviors. Genetic factors contribute to the incidence of ASD evidently. However, the genetic spectrum of ASD is highly heterogeneous. Chromosomal abnormalities contribute significantly to the genetic deficits of syndromic and non-syndromic ASD. In this study, we conducted karyotyping analysis in a sample of 500 patients (447 males, 53 females) with ASD from Taiwan, the largest cohort in Asia, to the best of our knowledge. We found three patients having sex chromosome aneuploidy, including two cases of 47, XXY and one case of 47, XYY. In addition, we detected a novel reciprocal chromosomal translocation between long arms of chromosomes 4 and 14, designated t(4;14)(q31.3;q24.1), in a patient with Asperger's disorder. This translocation was inherited from his unaffected father, suggesting it might not be pathogenic or it needs further hits to become pathogenic. In line with other studies, our study revealed that subjects with sex chromosomal aneuploidy are liable to neurodevelopmental disorders, including ASD, and conventional karyotyping analysis is still a useful tool in detecting chromosomal translocation in patients with ASD, given that array-based comparative genomic hybridization technology can provide better resolution in detecting copy number variations of genomic DNA. © 2013 Wiley Periodicals, Inc.
Autism is a childhood-onset complex neurodevelopmental disorder, characterized by the symptoms of impaired reciprocal social interactions, deficits in verbal communication, and the presence of restricted interests and/or stereotyped behavior. Broadly speaking, the patients with these behavioral problems were categorized as autism spectrum disorders (ASD), including autistic disorder, Asperger's disorder, and pervasive developmental disorders-not otherwise specified (PDD-NOS) according to the DSM-IV diagnostic criteria. The prevalence of ASD has dramatically increased to as high as approximately 9 and 11.3 per 1,000 in the US in most recent surveys in 2006 [report, 2009] and 2008 [report, 2012], respectively, and males are more often affected than females with a ratio of 4:1 [Kogan et al., 2009; Brugha et al., 2011; Chien et al., 2011b; Kim et al., 2011]. There is a large variation in the prevalence rates between studies [Brugha et al., 2011; Kim et al., 2011] explained by varied diagnostic and methodological approaches employed as well as different levels of awareness about ASD [Posserud et al., 2010]. Due to its high prevalence, long-term impairment, high genetic component, and lack of effective prevention and treatment, ASD has been prioritized for genetic studies [Merikangas and Risch, 2003].
Family and twin studies strongly indicate that the occurrence of ASD is genetically relevant, the heritability of autism was estimated about 90%, and ASD involves multiple genes [Li et al., 2012]. Besides genetic factors, the environmental factors also have some contributions [Ronald et al., 2011; Robinson et al., 2012]. The genetic architecture of autism is highly complex and heterogeneous. About 10% patients with ASD were comorbid with other syndromic disorders with known causative genetic defects, such as Rett's syndrome, fragile-X syndrome, tuberous sclerosis, Prader–Willi and Angelman syndromes, and Klinefelter syndrome [Veltman et al., 2005; Vincent et al., 2005; Jha et al., 2007; Young et al., 2008; Bishop et al., 2011]. However, the genetic causes of most idiopathic ASD remain unclear. Different approaches, such as linkage studies and association studies, have been undertaken to pin down the susceptible locus and genes associated with ASD without any consistent results [Hutcheson et al., 2003; Kim et al., 2006; Wang et al., 2011; Mosrati et al., 2012]. Conventional karyotype analysis showed that approximately 5–12% of ASD patients were associated with chromosomal structural aberrations [Li et al., 1993; Marshall et al., 2008]. Recently, using advanced array-based comparative genomic hybridization (array CGH) technology, more than 10% of patients with idiopathic ASD were found to have submicroscopic copy number variants (CNV) of genomic DNA [Ullmann et al., 2007; Marshall et al., 2008; Lo-Castro et al., 2009; Chien et al., 2010; Wisniowiecka-Kowalnik et al., 2010], suggesting genomic rearrangements contribute significantly to the genetic mechanism of autism. Although array CGH technology can help with identifying submicroscopic CNVs, conventional karyotyping analysis is still useful in detecting genomic rearrangements such as reciprocal translocation, inversion, and complex rearrangements [Hochstenbach et al., 2009]. In this study, as a part of our series of ASD genetic study we conducted a G-banding karyotype analysis in a large sample of patients diagnosed with ASD from Taiwan.
MATERIALS AND METHODS
We recruited 500 patients (447 males, 53 females), aged 3–17 (mean ± standard deviation, 9.08 ± 4.20) years old, who met the diagnostic criteria of autistic disorder or Asperger's disorder defined by the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) from the Department of Psychiatry, Taiwan University Hospital, Taipei, Taiwan; Department of Child Psychiatry, Chang Gung Memorial Hospital, Tao-Yuan and Kaohsiung, Taiwan; and Taoyuan Mental Hospital, Tao-Yuan, Taiwan, and special or resource education programs at nursery schools, kindergartens, primary, and high schools in northern Taiwan into our project of systematic genetic study of autism. The clinical diagnosis of autism was confirmed using the Chinese version of the Autism Diagnostic Interview-Revised by qualified child psychiatrists [Gau et al., 2010, 2011]. 93.8% of the patients with autism had abnormal development evident before 30 months of age based on the ADI-R interview and validated by the medical records. The subjects who were clinically or genetically diagnosed as Rett's syndrome, fragile X, or other chromosomal disorders or only met the diagnosis of PDD-NOS were excluded from this study.
The study protocol was approved by the Research Ethics Committee of all the study sites, written informed consents were obtained from the parents of patients and the patients if possible after the procedures were fully explained. All the 500 patients with autism were recruited for cytogenetic screening.
Karyotype analysis was performed on peripheral blood lymphocytes using the G-banding method according to the standard protocol established in the laboratory. Twenty metaphase cells were examined in each subject. The resolution of our protocol is around 500 bands on average.
Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA) was used for CNV analysis in this study. The SNP 6.0 array contains more than 1.8 million markers including more than 906,600 probes for SNPs and more than 946,000 probes for CNVs. These probes are evenly distributed across the whole genome with median distance between probes of ∼0.7 kb. The microarray experiment was conducted by the National Genotyping Center (Academia Sinica, Taipei, Taiwan) according the protocol provided by the manufacturer. CNVs were called by the computer program Genotyping Console 4.0 following the manufacturer's instructions (Affymetrix). Genes involved in the CNVs detected from this study were listed from the annotated gene symbols of the UCSC genome browser (NCBI36/hg 18).
A total of 500 patients with ASD, including 447 males and 53 females, received karyotyping analysis. Four patients were found to have gross chromosomal abnormalities, including two patients with 47, XXY (who had phenotype of Klinefelter syndrome), one patient with 47, XYY, and one patient with reciprocal translocation between the long arms of chromosomes 4 and 14, designated t(4;14)(q31.3;q24.1). The details of the clinical and experimental findings are given below.
Case 1 (U-1176)
This 9-year and 9-month-old boy is the second child of unrelated and healthy parents (Fig. 1A). He was found to have karyotype of 47, XXY in our laboratory. His sister, aged 17, performed very well at school. Despite no maternal infection or exposures to substances during the pregnancy, his mother suffered from vaginal bleeding in the first trimester and edema in the third trimester without increased blood pressure or blood sugar. She took multi-vitamines designed for pregnant woman from gestational age of 5–39 weeks. At his parents' ages of 38–39, he was born as a full-term baby weighted 3,980 g via natural delivery without any remarkable complications during infancy. His parents denied any systemic disease or neuropsychiatric disorders such as epilepsy later in life. He did not have any overt facial dysmorphic features but he was relatively taller than same-age peers (150 cm vs. 137.4 cm) with smaller hands, longer arms, overweight, bilateral small testes, and less masculine behaviors.
He walked alone at age 22 months old, and spoke his first word other than papa/mama at age 18 months old and first sentence after 3 years of age. At 2.5 years of age, he was clinically diagnosed with childhood autism based on DSM-IV and ICD-10 diagnostic criteria for autism by a board-certificated child psychiatrist. His major behavioral manifestations at age of 5 were: some non-meaningful verbal outputs mixed with self-talking in loud voice, immediate and delayed echolalia, pronoun reversal, a lack of social reciprocity, no direct gaze, no response to any verbal stimuli or gestures meant to catch his attention, self-stimulating behaviors like finger flicking, and self-spinning, ritual routine, a lack of tolerance to change, visual and tactile perceptional preoccupation, and stereotyped interest in number, alphabet, shape, and order.
Since the first psychiatric contact, he had received irregular but wide-ranging early intervention programs including occupational therapy, speech therapy, and educational assistance at preschool and kindergarten. In addition, he also had hyperactive symptoms that greatly interfered with behavioral therapy and education intervention but he has never been treated with any psychotropic agent.
At the age of 5 years and 4 months old, his mother was interviewed by using the Chinese ADI-R [Gau et al., 2011]. The clinical and ADI-R assessment clearly evidenced that the past and current symptoms of the patient reached the criteria for a childhood autism diagnosis as defined by the DSM-IV and ICD-10. The ADI-R revealed that in the past (current), the patient scored 21 (15) in the “qualitative abnormalities in reciprocal social interaction” area (cut-off = 10), 18 (15) in the “qualitative abnormalities in communication” (cut-off = 8), and 9 (9) in the “restricted, repetitive, stereotyped patterns of behaviors” (cut-off = 3).
No comorbid psychiatric disorder was noted based on parental interview with the Chinese version of the Kiddie Epidemiologic Version of the Schedule for Affective Disorders and Schizophrenia (K-SADS-E) [Gau et al., 2005]. The Wechsler Intelligence Scale for Children-3rd version (WISC-III) revealed a normal range of intelligence level (Full scale IQ, 103; Verbal IQ, 91; Performance IQ, 116). Currently, he maintained average academic performance but still showed overt social interaction deficits, inappropriate use of language, irrelevant speech with delayed echolalia, and preoccupied with number, routine, and rituals.
His mother and father also received clinical assessments and reported on the Adult Autism Spectrum Quotient (cut-off = 32) as 21 and 12, respectively [Lau et al., 2013]. Based on clinical assessment by the corresponding author (SSG), both his parents did not demonstrate any obvious autistic symptoms or DSM-IV psychiatric symptoms or disorders.
Case 2 (U-2148)
The patient, a 16-year-old boy, is the first child of unrelated and healthy parents and has a typically developing younger sister (Fig. 1B). He was found to have karyotype of 47, XXY in our laboratory. Regular blood tests and ultrasonic examinations during maternal pregnancy revealed negative findings. His mother reported that she did not expose to infection or substances such as coffee, or alcohol during the pregnancy; however, she may expose to 2nd-hand smoking from his father who smoked around 10 cigarettes per day at home. Despite no hypertension or increased blood sugar noted during pregnancy, his mother suffered from edema in the third trimester. She took multi-vitamins designed for pregnant woman during gestational age of 10–32 weeks. He was born through cesarean section smoothly due to maternal previous operation of uterus myoma, at both his parents' ages as 31 years old. He was a full-term baby with body weight of 3,450 g and received incubation care and phototherapy for seven days thereafter. His parents denied that he had any systemic disease or neuropsychiatric disorders except bilateral undescended testes. He received operation at age 7. He still suffered from small testes.
He walked alone at age of 18 months old, and spoke his first word other than papa/mama at the age of 1.5 years old and first sentence at his age of 2.5 years old. The patient was not found to have overt immediate echolalia but delayed echolalia and socially inappropriate and odd speech. His language is mainly used to express his basic needs and not for communication. However, his parents did not think patient had any abnormal socio-communication deficit at toddler stage until his age of 5 years old. He was then clinically diagnosed with Asperger's Disorder based on the DSM-IV and ICD-10 diagnostic criteria by a board-certificated child psychiatrist in a university hospital in Taipei. His major manifestations were: a lack of social reciprocity, no direct gaze, passive and inappropriate social interactions, delayed echolalia, socially inappropriate and stereotyped speech, and restricted and stereotyped behaviors and activities. He liked to draw cars and garages, bundle paper money, design questionnaire and ask schoolmates to complete it, do all statistics from school data and so on.
His interpersonal and socio-communication difficulties emerged over the first semester of Grade 1 at a primary school. At the age of 13.5 years old, his parents were interviewed by using the ADI-R [Gau et al., 2011] to confirm his diagnosis of Asperger's disorder. The ADI-R revealed that in the past (current), the patient scored 10 (6) in the “qualitative abnormalities in reciprocal social interaction” area (cut-off = 10), 8 (3) in the “qualitative abnormalities in communication” (cut-off = 8), and 7 (7) in the “restricted, repetitive, stereotyped patterns of behaviors” (cut-off = 3).
Assessed by using the Chinese K-SADS-E [Gau et al., 2005], the patient did not have any other psychiatric disorders than ASD. The WISC-III revealed a normal range of intelligence level (Full scale IQ, 97; Verbal IQ, 105; Performance IQ, 89). The Conners' Continuous Performance Test II (CCPT) and Wisconsin Card Sorting Test (WCST) did not reveal any abnormal neuropsychological function. Based on psychiatric and pediatric assessments, the patient had bigger breast, overweight, smaller testes, smaller hands, longer arms, feminine-like voice and behavior, and was taller (178 cm vs.169.8 cm) than adolescents of the same age.
His mother and father also received clinical assessments and reported on the Adult Autism Spectrum Quotient (cut-off = 32) as 28 and 22, respectively [Lau et al., 2013] revealing neither obvious autistic symptoms nor DSM-IV psychiatric disorders except that his father suffered from some anxious, obsessive, and impulsive symptoms.
Case 3 (U-2875)
This 9-year and 7-month-old boy is the only child of unrelated and healthy parents (Fig. 1C). He was found to have karyotype of 47, XYY in our laboratory. Maternal pregnancy history did not reveal any maternal infection or exposures to substances, and regular laboratory examinations did reveal any abnormal findings. However, amniocentesis revealed abnormal chromosome results (47, XYY). She took multi-vitamines designed for pregnant woman from gestational age of 20–40 weeks. The patient was born through cesarean section due to dystocia, at his father's age of 49 and mother's age 46. He was a full-term baby with body weight of 3,500 g without any remarkable complications during post-natal and infancy. His parents denied any systemic disease or neuropsychiatric disorders except one episode of febrile convulsion. He did not have any minor physical anomalies.
He walked alone and spoke his first word other than papa/mama at age of 12 months old and first sentence at age of 18 months old. He demonstrated his superior role memory in number and Chinese words at preschool age. Although he can understand and follow simple orders, he had limited comprehension to conversation and a lack of socially appropriate communication. At his age of 6.5 years old, he was clinically diagnosed with Asperger's Disorder based on DSM-IV diagnostic criteria by a board-certificated child psychiatrist in middle Taiwan. He and his family then moved to Taipei and he started to receive short-term treatment of occupational therapy to improve his motor coordination at a community hospital in Taipei.
In addition to his special interest in reading science books and preoccupied with number, cars, maps, and rules of games he like, he was found to have difficulty understanding what others thought about themselves and him, inappropriate affective expression, impaired social reciprocity, and a lack of socially appropriate communication. He liked to draw cars, streets, bridges, and maps in a repeated and stereotyped way, and talked to others even strangers in loud voice about his science study without considering whether his conversation was interesting to them. He was easily frustrated and became irritable if he was not paid attention on his requests of special rules and activities. He always carried the same puppet with him everywhere. He was very sensitive to noise and sought satisfaction from smelling and touching objects.
At the age of 8 years and 11 months old, his parents were interviewed by using the Chinese ADI-R [Gau et al., 2011]. The ADI-R revealed that in the past (current), the patient scored 14 (7) in the “qualitative abnormalities in reciprocal social interaction” area (cut-off = 10), 12 (11) in the “qualitative abnormalities in communication” (cut-off = 8), and 9 (7) in the “restricted, repetitive, stereotyped patterns of behaviors” (cut-off = 3). His autistic symptoms were clearly observed before 3 years old.
The Chinese K-SADS-E [Gau et al., 2005] assessments revealed some hyperactivity and oppositional symptoms without reaching the diagnosis of ADHD or oppositional defiant disorder. The WISC-III revealed a superior intelligence level (Full scale IQ, 120; Verbal IQ, 117; Performance IQ, 120). The CCPT revealed poorer vigilance but the WCST did not reveal any abnormal set-shifting ability. His performance on the Raven's Progressive Matrices reached 98 percentages, suggesting excellent visuo-spatial ability. Currently, he maintained the first place in academic performance in his class and showed low frustration tolerance if he did not get the first place.
His mother and father reported on the Adult Autism Spectrum Quotient (cut-off = 32) as 23 and 8, respectively [Lau et al., 2013] and received clinical assessments by the corresponding author (SSG) showing no overt psychopathology, and some autistic-like symptoms and stereotyped life style, respectively.
Case 4 (U-1344)
This 18-year-old young man is the only child of unrelated and healthy parents (Fig. 1D). Although regular blood tests and ultrasonic examinations during maternal pregnancy did not find any abnormal results, amniocentesis revealed chromosome 4 and 14 translocation from father's side. Mother's attending physician suggested that patient should be a normal baby because his father had the same chromosomal deficits. The result was confirmed by our laboratory showing translocation between 14q24.1 and 4q31.1 (Fig. 2), and CNV analysis using Affymetrix SNP 6.0 platform did not reveal apparent gain or loss of DNA segments associated with the translocation. Nevertheless, we identified a total of 8 CNVs in this patient. Notably, we found a microduplication of about 0.73 Mb located at Xp22.32 that disrupted the neuroligin 4, X-linked gene (NLGN4X). The mother denied any infection or exposures to substances such as coffee, alcohol, or tobacco during her pregnancy but took multi-vitamines designed for pregnant woman during gestational age of 3–39 weeks. At his parents' age of 38 years old, he was born as a full-term baby through cesarean section due to premature rapture of membrane and prolonged labor with fetal distress for three times. His baby birth weight was 3,264 g and was kept in incubator and received phototherapy due to prolonged jaundice for 5 days. His parents denied any systemic disease or neuropsychiatric disorders such as epilepsy later in life. He did not have any dysmorphic features.
He walked alone at age of 12 months old, and spoke his first word other than papa/mama at 27 months of age and first sentence at 36 months of age. The patient's language was mainly used to express his basic needs and not for communication. At 13 years of age, he was then clinically diagnosed with Asperger's Disorder by the corresponding author. The major manifestations were: passive and socially-inappropriate interpersonal interactions and conventional/instrumental gestures, social disinhibition, impaired direct gaze, delayed echolalia, socially inappropriate and stereotyped speech, and restricted and stereotyped behaviors and activities. His circumscribed interest was insects and only talked to others about insects. He was easily frustrated and became irritable if his requests of special rules and activities were not accepted or paid enough attention.
In addition to autistic core symptoms as mentioned above, he was found to have extreme hyperactivity, impulsivity, and inattention since his age of 3. These symptoms had apparently impeded the progress of early intervention for his language difficulties and poor emotional and behavioral controls. He was clinically diagnosed with ADHD at 8 years of age. At 13-year and 5-month old, he started to take methylphenidate till now (last daily dose, immediate-release methylphenidate, Ritalin, 20 mg).
The Chinese ADI-R interview [Gau et al., 2011] at his age of 13 revealed that in the past (current), the patient scored 19 (10) in the “qualitative abnormalities in reciprocal social interaction” area (cut-off = 10), 13 (7) in the “qualitative abnormalities in communication” (cut-off = 8), and 8 (5) in the “restricted, repetitive, stereotyped patterns of behaviors” (cut-off = 3).
His ADHD symptoms assessed in 2008 at his age of 14 based on the Chinese K-SADS-E interview [Gau et al., 2005] confirmed his clinical diagnosis of ADHD. His mother also reported on the Chinese version of the Swanson, Nolan, and Pelham, version IV scale [Gau et al., 2008] about the patient, which revealed nine items of inattention and three items of hyperacitivity-impulsivity based on the DSM-IV ADHD symptom criteria.
The WISC-III revealed his intelligence within the lower level of normal range (verbal IQ, 91; performance IQ, 78; full-scale IQ, 83). The CCPT did not revealed apparent attention difficulty; whereas the WCST revealed more total errors, perseverative response errors and non-perseverative errors and lower conceptual level responses than typically developing children of the same age, suggesting impaired cognitive flexibility in the patient. Currently, he maintained below academic performance at a senior high school and showed abnormal social reciprocity, socially-inappropriate communication, stereotyped behaviors/speech, and preoccupied with his special interests and ritual behaviors.
His mother and father reported on the Adult Autism Spectrum Quotient (cut-off = 32) as 19 and 18, respectively [Lau et al., 2013]. Based on clinical assessment by the corresponding author and their reports about themselves on the Chinese version of the Adult Self Report Inventory-4 [Chien et al., 2011a], both his parents did not demonstrate any obvious autistic symptoms or DSM-IV psychiatric symptoms or disorders.
In this study, we conducted karyotyping analysis in a sample of 500 patients with ASD from Taiwan. To the best of our knowledge, this is the largest sample of such kind of study in Asia. In a previous karyotyping analysis of patients with autism from Taiwan, Li et al.  reported that 12 out of 104 patients had chromosomal abnormalities, including eight patients with fragile X syndrome, two patients with Down syndrome, one patient with reciprocal translocation between chromosomes 5 and 6, and one patient with Y inversion. There are apparently different findings between their study and ours. The most frequent genetic deficiency reported in ASDs were Fragile X syndrome, tuberous sclerosis, Rett's syndrome, Prader–Willi and Angelman syndrome; the prevalence of each disorders in ASD was in the range of 1–2% [Devlin and Scherer, 2012], suggesting the genetic basis of ASD is highly heterogeneous. In our studying cohort, we have excluded the subjects who were clinically or genetically assessed to have apparent Down's syndrome, Fragile X syndrome or Rett's syndrome in order to discover the unknown chromosome defects associated with ASD, especially those with chromosomal translocation. That may in part explain the different findings in these two studies.
Three patients with sex chromosome aneuploidy were diagnosed as Asperger's disorder. Two patients with 47, XXY that is also named Klinefelter syndrome, is the most common sex chromosome disorder in males, affecting one in 660 men [Groth et al., 2013]. In addition to its various physical and physiological abnormalities, patients with Klinefelter syndrome have high psychiatric comorbidity, behavioral problems, and cognitive impairments [Leggett et al., 2010]. In a review paper, Savic reported that the behavioral phenotype of Klinefelter syndrome is characterized by language, executive and psychomotor dysfunction, as well as socioemotional impairment. The prevalence of schizophrenia, attention deficit hyperactivity disorder, ASD and affective regulation problems in patients with Klinefelter syndrome also increased [Savic, 2012]. Notably, the increased vulnerability for ASD and features in Klinefelter syndrome have been reported by several studies [Jha et al., 2007; van Rijn et al., 2012, 2008; Tartaglia et al., 2010; Bishop et al., 2011; van Rijn and Swaab, 2011; Ross et al., 2012]. Similar to Klinefelter syndrome, patients with XYY syndrome are also vulnerable to ASD, which has been reported by several studies [Gillberg et al., 1984; Nicolson et al., 1998; Kielinen et al., 2004; Bishop et al., 2011; Ross et al., 2012]. Thus, elucidation of molecular mechanism of the association of Klinefelter syndrome and XYY and ASD should shed some light on the pathogenesis of autism. In a recent study, Bryant and colleagues reported that patients with XYY had increased total white and gray matter volumes of brain, a finding putatively related to the increased frequency of ASD in individuals with this condition. They also found frontotemporal gray and white matter reductions in XYY syndrome, which provides a likely neuroanatomical correlate for observed language impairments [Bryant et al., 2012]. Bishop and Scerif also proposed that Klinefelter syndrome as a window on the etiology of language and communication impairments in children, as sex chromosomes contain the neuroligin genes that are associated with specific language impairments [Bishop et al., 2011].
In this study, we also identified a patient carrying a reciprocal translocation between chromosomes 1 and 4. Given its rare frequency, chromosomal translocation provides an opportunity to identify pathogenic genes of psychiatric disorders if the translocation cosegregates with mental illness. The well-documented example is the discovery of the DISC1 gene associated with schizophrenia. The gene was disrupted by a reciprocal translocation between chromosomes 1 and 11 that cosegrgated with various psychiatric disorders in a large Scottish pedigree [Porteous et al., 2011]. Similar to DISC1 in schizophrenia, several genes associated with ASD were also identified through mapping the breakpoints of chromosomal translocation found in patients with ASD. For example, ZF407 [Ren et al., 2013], GNB1L [Chen et al., 2012], and AUTS2 [Huang et al., 2010] were all identified from balanced translocation found in patients with autism. In this study, we identified a balanced reciprocal translocation t(4;14)(q31.3;q24.1) in a patient with ASD. This translocation is a novel one, not reported in the literature. Nevertheless, the balanced translocation was inherited from the patient's asymptomatic father, suggesting this translocation may not be pathogenic and not associated with ASD. Other possible explanation may include incomplete penetrance or varied expressivity of this translocation, or the translocation needs interactions with environmental factors or other genetic mutation in order have clinical manifestation [Gau et al., 2012]. Given its uncertain association with ASD in this patient, it is still worthwhile to map the breakpoints of the translocation to identify genes that may be implied in autism genetics in future study. In this patient, we also identified a microduplication at chromosome Xp22.32, in which the NLGN4X was disrupted by the microduplication. As mutations in the NLGN4X gene have been reported to be associated with autism or mental retardation in several studies [Jamain et al., 2003; Laumonnier et al., 2004; Ylisaukko-oja et al., 2005; Pampanos et al., 2009; Yanagi et al., 2012], it is likely that the psychiatric symptoms of this patient might be related to this microduplication. Nevertheless, a patient with a deletion involving NLGN4X was found to have normal intelligence and social interactions [Mochel et al., 2008]. Hence, further research is needed to clarify the genetic basis of this patient.
Genetic screening approach is a reliable way to discover the genetic factors associated with ASD, many reports have demonstrated their achievements [Miles, 2011]. We have successfully identified the chromosome abnormalities carried by the patients of ASD using G-banding karyotype screening. Although, not many patients who carry chromosome aberrations were found in our study, the information we obtained is valuable to improve our understanding of ASD. The scanty findings of chromosome abnormalities may reflect that there are still many hidden genetic factors associated with idiopathic autism waiting to be identified. Applying multiple genetic screening technologies to the same studying cohort can improve the detecting rate of risk genetic factors. Recently, the next generation sequencing technology is a powerful tool in identifying the disease-associated mutations. The high throughput, high resolution and multi-application features of this technology can help to speed up the genetic screening in ASD research as well [Alkan et al., 2011]. However, it is still a resources-consuming technology and needs a lot of bioinformatic efforts to make the results comprehensive. Before we can switch our research energy to the advanced next generation sequencing, using the well-established methods for genetic screening is an efficient way under limited resources. The relationship between ASD and copy number variations (CNVs) has been intensively addressed, using well-established array-based CGH to screen for the submicroscopic copy number changes in our patients of ASD will be the next stage. Benefited from our large sample size and unabridged clinical interview, we anticipate a significant conclusion in our further studies.