Molecular and clinical characterization of Angelman syndrome in Chinese patients

Transcription

1 Clin Genet 2014: 85: Printed in Singapore. All rights reserved Short Report 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Molecular and clinical characterization of Angelman syndrome in Chinese patients Bai J.-L., Qu Y.-J., Jin Y.-W., Wang H., Yang Y.-L., Jiang Y.-W., Yang X.-Y., Zou L.-P., Song F. Molecular and clinical characterization of Angelman syndrome in Chinese patients. Clin Genet 2014: 85: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2013 Angelman syndrome (AS) is a neurobehavioral disorder caused by lack of function of the maternal copy of the ubiquitin-protein ligase E3A (UBE3A) gene. In our study, 49 unrelated patients with classic AS phenotypes were confirmed by methylation-specific PCR (MS-PCR) analysis, short tandem repeat linkage analysis, and mutation screening of the UBE3A gene. Among the Chinese AS patients, 83.7% (41/49) had deletions on maternal chromosome 15q Paternal uniparental disomy, imprinting defects, and UBE3A gene mutations each accounted for 4.1% (2/49). Two AS patients were confirmed by MS-PCR analysis, but the pathogenic mechanism was unknown because their parents samples were unavailable. Of the two described UBE3A gene mutations, that is, p.pro400his (c.1199c>a) and p.asp563gly (c.1688a>g), the latter has not been reported previously. Mutation transmission analysis showed that the p.pro400his and p.asp563gly mutations originated from asymptomatic mothers. The patients with the maternal deletion showed AS clinical manifestations that were consistent with other studies. However, the incidence of microcephaly (36.7%, 11/30) was lower than that in the Caucasian population (approximately 80%), but similar to that of the Japanese population (34.5%). Our study demonstrated that the occurrence of microcephaly in AS may vary among different populations. Conflictofinterest All the authors have declared no potential conflicts of interests. J.-L. Bai a,y.-j.qu a,y.-w.jin a, H. Wang a, Y.-L. Yang b,y.-w. Jiang b, X.-Y. Yang c, L.-P. Zou d and F. Song a a Department of Medical Genetics, Capital Institute of Pediatrics, Beijing, , China, b Department of Pediatrics, Peking University First Hospital, Beijing, , China, c Department of Neurology, Beijing Children s Hospital, Capital Medical University, Beijing, , China, and d Department of Pediatrics, General Hospital of Chinese People s Liberation Army, Beijing, , China Key words: Angelman syndrome clinical characterization genetic analysis pathogenic mechanism ubiquitin-protein ligase E3A Corresponding authors: Fang Song, Department of Medical Genetics, Capital Institute of Pediatrics, Beijing , China. Tel.: ; fax: ; songf_558@263.net and Li-Ping Zou, Department of Pediatrics, General Hospital of Chinese People s Liberation Army, Beijing , China. Tel.: ; fax: ; zouliping21@sina.com Received 23 October 2012, revised and accepted for publication 27 March 2013 Angelman syndrome (AS) is a neurodevelopmental disorder caused by lack of function of the maternal UBE3A at 15q11-q13, characterized by intellectual disability, severely delayed speech, balance disorder, and characteristic behaviors (1). Seizures and abnormal electroencephalograms (EEGs) are often observed in AS patients. The incidence of AS is estimated to be 1 in 15,000 (2). To date, four known mechanisms have been implicated in AS: maternal deletion of 15q11-q13, paternal uniparental disomy (UPD), imprinting defects, and mutations in the UBE3A gene (3). These defects affect the maternal expression of UBE3A and disturb the combination of the E6-AP protein and specific substrates, which leads to the phenotype of AS (4). The UBE3A gene comprises 16 exons that span approximately 120 kb. Exons 7 16 are coding exons for UBE3A (5). UBE3A encodes E6-AP ubiquitinprotein ligase, which is biallelically expressed in most tissues (6), but maternally expressed in the neurons 273

2 Bai et al. (7). UBE3A protein is characterized by a conserved C-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain, which contains 350 amino acids. The HECT domain plays an important role in substrate recognition and subsequent degradation of target proteins (3). In this study, we sought to perform genetic analyses of AS in Chinese patients and to describe genotype phenotype correlations in these patients. Materials and methods Subjects A total of 49 unrelated Chinese AS patients, whose clinical diagnoses had been made according to the updated consensus for diagnostic criteria (1), were enrolled in this study. Informed consent was obtained from the patients parents. This project was approved by the ethics committee of Capital Institute of Pediatrics. DNA methylation analysis Genomic DNA was extracted using phenol chloroform extraction. Duplex MS-PCR was used to analyze the methylation status of SNRPN gene. The sequences of specific primers and PCR conditions were as described by Kosaki et al. (8). Short-tandem repeat (STR) analysis Six STR markers from regions inside and outside of the 15q locus were selected for linkage analysis (9). The primers were as indicated in the UCSC database. Mutation analysis for UBE3A Exons 7 16 and their intron exon flanking sequences of the UBE3A gene were described by Fang et al. (5). For confirmed mutations, we performed experiments to exclude the possibility of being a polymorphism variation. For the p.asp563gly (c.1688a>g) mutation, sequence analysis of exon 10 was performed in 12 individuals of the 43 maternal relatives of 5 generations. Clinical records Medical examinations were performed and related questionnaires were developed according to the AS updated consensus for diagnostic criteria (1). Microcephaly was defined as head growth measuring below the third centile for age and sex or 2 SD below the mean of the growth standardized chart of Chinese children (10). The contents of the questionnaires included development of gross motor skills (age of head control, sitting unsupported, and walking independently), language performance, typical AS clinical manifestation, auxiliary examinations (EEG monitoring, cranial magnetic resonance imaging [MRI]/computed tomography [CT], and metabolic screening tests), and family history. Statistical analysis The proportions of clinical characteristics in subgroups of patients were compared using Fisher s exact test, which generates exact probabilities corresponding to the null hypothesis of nonassociation. Results Genetic studies Of 49 patients, 47 showed AS abnormal methylation patterns in the 15q11-q13 region with the presence of the paternal allele, but the absence of the maternal allele. For 45 of the 47 patients, we performed linkage analysis. The result of linkage analysis revealed that 41 patients had maternal deletions and 2 patients had paternal UPD. For two patients with abnormal methylation patterns, but normal results of STR analysis, we reasoned that the imprint defect may be present; however, we were unable to confirm this hypothesis. The remaining two AS patients with negative results in MS-PCR were screened for mutations in the coding sequence of the UBE3A gene by the Sanger sequencing method. Two missense mutations, p.pro400his and p.asp563gly, were identified in two unrelated patients who were inherited maternally. The p.pro400his has been reported previously as a causative mutation for AS (9). The p.asp563gly mutation located in exon 10 of the UBE3A gene is novel and has not been reported previously. The Asp563 residue was located in the conserved HECT domain and was evolutionarily conserved across many species, including Homo sapiens, Mus spretus, Mus musculus, Danio rerio, Xenopus tropicalis, Laevis xenopus, and Bos taurus. Sequence analysis of the p.asp563gly mutation in extended family members revealed that the maternal grandfather carried the same sequence change (c.1688a>g), consistent with the conclusion of maternal imprinting. Sequence analysis of other normal family members did not reveal any positive findings that support the sequence being a pathologic mutation (Figs 1 and 2). Clinical manifestations in Chinese AS patients Patients with UBE3A mutations Clinical manifestations of AS in the patient with the p.asp563gly mutation included intellectual disability, language delay, movement disorder, and behavioral features, but no seizures or microcephaly. The patient could speak 4 5 words and count 0 5 numbers after receiving extensive speech therapy for 7 years (from the age of 1.5 to 9 years) despite a significant problem in pronunciation. The patient s receptive language was much better than his expressive language, which may be attributed to the continuous rehabilitation training in speech or may have resulted from the milder effect of the mutation on language function. Compared to the patient with the p.asp563gly mutation, the patient with the p.pro400his mutation had 274

3 Molecular and clinical characterization of Angelman syndrome Fig. 1. DNA sequence chromatograms of the p.asp563gly in UBE3A from the patient and his parents. Note: p.asp563gly was detected in the patient and his mother, but not in his father. The underlines mark codon 563. Fig. 2. Pedigree of the AS case with respect to the p.asp563gly mutation. The patient, his asymptomatic mother, and grandfather carried the same mutation (p.asp563gly). However, other relatives of the patient s mother were negative for the mutation. Note: a star( ) marks the members who were screened for the mutation. typical, more severe AS phenotypes. Besides common motor developmental delays and a happy disposition, the patient also had microcephaly, absence of speech, abnormal EEG, and seizures. Patients with maternal deletion Of 41 patients with maternal deletions, 30 patients with sufficient clinical data (15 boys and 15 girls) were analyzed (Table 1). The mean age at diagnosis was 27.3 ± 13.3 months (range: 8 60 months), and the mean age at the last visit was 5.3 ± 2.1 years (range: years). Overall, the clinical features of these AS patients were consistent with those reported in the literature. Gross motor development was delayed in these patients. The mean ages for acquisition of head control and sitting independently were approximately 5.2 ± 2.1 months (range: 4 12 months) and 12.4 ± 5.1 months (range: 7 24 months), respectively. The mean age for patients to start walking (n = 24) was 33 ± 7.1 months (range: months). Twentyseven patients had a vocabulary of only one or two words. Other clinical manifestations, such as protruding tongue, prognathia, frequent drooling, and strabismus were seen in 40 80% of patients. Approximately 80% of the patients presented with flat occiput, wide mouth, wide-spaced teeth, hypopigmented skin, and sleeping disturbance. Approximately 90% of the patients had abnormal EEGs, such as high amplitude rhythmic 2 3/s spike-wave activity. MRI or CT analysis revealed that cortical atrophy or dysmyelination was observed in 60% of the patients. Microcephaly is a common feature reported in more than 80% of AS patients in the Caucasian population. Interestingly, only 36.7% (11/30) of patients in our cohort had a head circumference (HC) less than the third centile (36.7%), meeting the clinical diagnosis of microcephaly. The incidence of microcephaly was much lower in our study than that in the Caucasian population, but the cause underlying this difference was not immediately clear. It should be pointed that a similar reduced occurrence of microcephaly (34.5%) was also observed in Japanese AS patients. For the microcephaly group and non-microcephaly group, with the exception of higher proportions of flat occiput in the microcephaly group (p = 0.02 and <0.05), there were no significant differences in other clinical features (p > 0.05). Discussion Our study revealed the similar distribution of known pathogenic mechanisms in 49 Chinese AS patients with that in another study (11). In our study, UBE3A gene mutations, p.asp563gly and p.pro400his, were found. p.asp563gly is a novel mutation and is located in the HECT domain. Thus, it may result in the loss of function of E6-AP by influencing substrate recognition and ubiquitin transfer (3). Mutation analysis confirmed that the mutation was inherited from this proband s grandfather through his asymptomatic mother. The siblings of the proband s grandfather did not carry the mutation, implying that the mutation may have originated from spermatogenesis in the great grandfather. Therefore, the inheritance pattern was consistent with known maternal imprinting patterns for AS in the UBE3A gene. The patient with the p.asp563gly mutation presented milder clinical phenotypes compared to AS patients with maternal deletions in terms of development, particularly in speech, which may have been related to the continuous rehabilitation training in speech. The effects of the 275

4 Bai et al. Table 1. Clinical features of sporadic AS patients with maternal deletion Clinical manifestations Our study Saitoh (1994; Japan) MD group NMD group Total SD cases Number % Number % Number % Number % Head control 11/ / / SI 11/ / / WI 8/ / /30 80 Mental retardation 11/ / / / Ataxic movement 8/ / / / Speech impairment 11/ / / / Laughing and smiling 11/ / / / Epilepsy 11/ / / / Abnormal EEG 10/ / / / Flat occiput 11/ / / / Macrostomia 8/ / / /35 80 Prognathism 8/ / / /35 80 Frequent drooling 9/ / / Protruding tongue 6/ / / / Hypopigmented skin 10/ / / / Feeding difficulty a 6/ / / Sleeping disorder 9/ / / Strabismus 7/ / / / Abnormal MRI b 7/ / / MD group, microcephaly in deletion group; NMD group, non-microcephaly in deletion group; SD cases, sporadic deletion cases; SI, sitting independently; WI, walking independently; : not available. a Indicated feeding difficulty as infant. b Indicated abnormal MRI showing atrophy or delayed myelination. p.asp563gly mutation on language functions will need to be further studied. In our study, the patients were obviously delayed in gross movement. The mean age for sitting without support was approximately 12 months, similar to that in another study (12). For the 24 patients who could walk, the mean age of walking independently was 33 months, which was earlier than that reported in another study (12). The proportion of our patients with seizures was approximately 90%, which was higher than that reported by Tan et al. (71%), but similar to that reported by Smith et al. (96%), Varela et al. (89.4%), and Saitoh et al. (100%) (13 16). The cohorts of our study and three other studies were all over age 5 years, which was older than that of the cohort studied by Tan et al. (<5 years) (16). This may account, in part or in whole, for the difference in the prevalence of seizures. Approximately 80% of our patients had hypopigmented skin, which was similar to that of Japanese AS patients (88%), but higher than that reported by Tan et al. (39%). This difference in the rate may be related to the skin color differences between Asian and Caucasian populations or may be attributed to different mutations in the P gene located in the remaining paternal chromosome. Sleeping disturbance was also observed in more than 80% of our patients, indicating that sleeping disorder is a common and characteristic behavior of AS deletion patients. Approximately 90% of patients had characteristic EEGs, indicating that EEGs are important for AS diagnosis. Notably, microcephaly was only observed in 36.7% (11/30) of our AS cohort, which was lower than that in the Caucasian population (1, 16), but similar to that in Japanese AS patients (14). This observation is interesting, although the cause for this difference is not immediately clear. To our knowledge, this is the largest molecular and clinical study of AS in Chinese population to date. The patterns of molecular mechanisms underlying AS were comparable to those in the Caucasian population. Although there was little difference in motor and speech development as well as seizure frequency compared to other studies, the frequency of microcephaly was much lower in the Chinese cohort. This observation implied that normal HC in children with significant developmental delays may not exclude AS in the Chinese population. Understanding the cause of these differences in the two populations may provide insights into the elucidation of the molecular mechanisms contributing to microcephaly due to the deficiency in UBE3A protein. Acknowledgements We would like to thank the patients and their families for their participation. We are grateful to Professor Tong Ming For for technical assistance. We would like to express our gratitude to Professor Yong-Hui Jiang for critical reading of the manuscript. This work was funded in part by the Science Foundation of Capital Institute of Pediatrics (Proj. No: 2007-A01). 276

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