The Clinical and Molecular Spectrum of 15q Duplication Syndrome in Chinese
15q duplication syndrome (OMIM #608636) is a neurodevelopmental disease that characterised by hypotonia, developmental delay, intellectual disability, epilepsy and distinctive facial gestalt. A territory-wide study of 15q duplication syndrome is performed in Hong Kong with aim to examine its clinical and molecular features among Chinese patients. There are total of 12 cytogenetically and molecularly confirmed individuals between the period of January 2011 and December 2015. Four of them have interstitial duplication and 8 of them have isodicentric chromosome 15. The prevalence of 15q duplication syndrome in our Chinese cohort with intellectual disability and autistic spectrum disease is estimated to be 1.0% and 2.9%, respectively. As compared with western population, epilepsy is less common while squint is more prevalent in our Chinese patients. However, no genotype-phenotype correlation can be demonstrated in this study. Conclusion: The prevalence and clinical features of 15q duplication syndrome patients in Hong Kong Chinese are comparable with other western populations. It is hope that by having the better understanding of its underlying pathomechanism and their genotype-phenotype correlation would lead to better management and genetic counselling for patient with 15q duplication syndrome.
Keyword : 15q duplication syndrome; Chinese; Interstitial duplication 15q11-q13; Isodicentric chromosome 15
The chromosome 15q11-q13 region is a complex imprinted region that prone to genomic rearrangement. Within this region, some genes are only expressed on the maternally inherited chromosome 15, like UBE3A and ATP10C; while other genes are only expressed on the paternally inherited chromosome 15, like MKRN3, MAGEL2, NDN, C15orf2, SNURF-SNRPN. Moreover, this region harbours five duplicons or breakpoints (BP) that consisted of large segment low copy repeats (LCR).1,2 Through non-allelic homologous recombination and U-type crossover mechanism,3 various forms of deletion, duplications, triplications, translocations, and supernumerary marker chromosomes (SMC) at proximal chromosome 15q region would result. Depend on the parent-of-origin and the size of rearrangement, such genomic rearrangement would lead to Angelman syndrome, Prader-Willi syndrome and 15q duplication syndrome.
15q duplication syndrome (OMIM #608636) is a clinically recognisable neurogenetic syndrome. It is caused by either interstitial duplication of chromosome 15q11.2 or extra isodicentric chromosome 15(idic(15)(p11.2-13.3)).3 The clinical features included hypotonia, developmental delay, intellectual disability, epilepsy and distinctive facial gestalt. Most affected individuals would meet the diagnostic criteria of autism or autism spectrum disease. Apart from that, some would also develop behavioural problems like anxiety disorder and attention deficit hyperactivity disorder.3,4 The prevalence of 15q duplication syndrome in autism cohort is estimated to be 1-3% in early studies.5,6 However, the prevalence, comprehensive clinical spectrum and molecular study of 15q duplication syndrome have never been reported in Chinese.
The aim of this study is to summarise the clinical and genetic findings of all cytogenetically and molecularly confirmed 15q duplication syndrome patients in Hong Kong Chinese.
Patients and Method
The Clinical Genetic Service (CGS) of Department of Health is the only government funded tertiary genetic referral center that provides comprehensive genetic counselling, diagnostic and laboratory service for the whole of Hong Kong population. More than 95% of population in Hong Kong is ethnic Chinese. Patients with suspected genetic or syndromic cause of intellectual disability and autism or autism spectrum disease (ASD) are referred for clinical assessment and genetic testing.
In this study, all records of patients with genetically confirmed 15q duplication syndrome between January 2011 and December 2015 under CGS are retrieved from the computer database system. The clinical and laboratory data of these patients are being analysed. The diagnosis of intellectual disability, autism and autistic spectrum disease are made by paediatricians, developmental paediatricians, clinical psychologists or child psychiatrists.
Cytogenetic and Molecular Study
The diagnosis of 15q duplication syndrome is confirmed either by standard cytogenetic G banding technique with fluorescence in situ hybridization (FISH) method or array Comparative Genomic Hybridization (aCGH) study. Further molecular studies like microsatellite analysis with parental DNA or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) is used to delineate the parent-of-origin of the 15q duplication.
Patients are screened for rearrangements involving the 15q11-q13 region with the ME028 PWS/AS (MRC-Holland, Amsterdam, The Netherlands). The copy number change and methylation status is detected by methylation-sensitive restriction enzyme. Analysis of the MS-MLPA PCR products is performed on an ABI3500 Genetic Analyser using the GeneMapper software (Applied Biosystems, Foster City, Calif., USA). For copy number analysis, the data generated are being intra-normalised by dividing the peak area of each amplification product by the total area of the reference probes. The ratios are then obtained by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference runs. For methylation analysis, the intra-normalised peak area of each MS-MLPA probe from the digested sample is divided by the value obtained for the undigested sample.
Fluorescence In Situ Hybridization (FISH)
FISH is performed on metaphase chromosome spreads preparations from peripheral blood. Three commercial DNA probes that hybridize to the SNRPN locus at 15q11q13 region, PML locus at 15q22 region and centromere of chromosome 15 at 15p11.2 region from Vysis (Abbott) are being used. Ten metaphase cells are being examined to detect cells with loss or gain of specific signals.
The microsatellite analysis is studied by using standard protocol. Haplotype analysis is being performed by using eleven polymorphic markers in 15q regions (D15S219, Gabrb3, SC2, 14150, D15S217, M37, SC3, AFM262, AFM323, AFM291 and AFM164). PCR products are visualised with a DNA sequencer and allele sizes are determined by using Genescan and Genotyper software (Applied Biosystems). A positive diagnosis required evidence of unique parental inheritance at ≥informative 2 markers.
High-resolution whole genome analysis is performed by using PerkinElmer CGXTM 8x60K Human Genome microarrays (Agilent Technologies, Santa Clara, CA). This contains approximately 60 000 sixty-mer probes with resolution of 190Kb in the backbone and 28kb average probe spacing. Labelling is performed by using Agilent Genomic DNA enzymatic labelling kit (Agilent Technologies, Santa Clara, CA, USA) and clean-up of labelled genomic DNA is performed by using Amicon ultra 0.5 ml centrifugal filters according to manufacturers' instructions (Millipore, Billerica, MA, USA). Slides are scanned on an Agilent scanner and processed with Feature Extraction software (v10.7). Each patient DNA is labelled in Cy5 and sex matched reference DNA is labelled in Cy3, which then co-hybridized against the array chip. Results are analysed by using Agilent Genomic Workbench (v6.0 and v6.5) software with the following settings: ADM2 as aberration algorithm, threshold of 6.0, moving average 2 Mb. The results are according to Human Genome build 19 and include imbalances with at least three consecutive probes with abnormal log2 ratios. All the imbalances are interpreted by consulting the UCSC genome browser (http://genome.ucsc.edu), Decipher (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources - http://decipher.sanger.ac.uk/), ClinGen (Clinical Genome Resource - https://www.clinicalgenome.org/), OMIM (Online Mendelian Inheritance in Man - http://www.ncbi.nlm.nih.gov).
Data Analysis and Statistical Calculation
Epidemiological data, physical characteristics, growth records and molecular findings are then collected for analysis. Clinical photographs are taken during consultation with written consent. For statistical calculation, Fisher's exact test is used for categorical variables. Two-tailed p-values are being computed. Differences are considered to be statistically significant when the p-value is ≤0.05.
During this 5 years' study period, a total of 1,116 patients with intellectual disability and 313 patients with autism or ASD are being referred to our genetic clinic for assessment. Twelve individuals among 10 families with 15q duplication syndrome are diagnosed. All are ethnic Chinese. Their current age in year 2016 are ranged from 3 years 6 months to 34 years old, with a median of 8 years 2 months old. The male to female ratio was 1:2.
Among them, 11 of them have intellectual disability and 9 of them have autism or ASD. Therefore the prevalence of 15q duplication syndrome in our Chinese cohort with intellectual disability and autism or ASD is 1.0% and 2.9%, respectively.
Concerning the underlying genetic mechanism, 4 of them have interstitial duplication and 8 have isodicentric chromosome 15. Two out of these 8 isodicentric cases are in mosaic pattern with abnormal cell lineage ranged from 50% to 93%. The genomic aberration for all symptomatic cases are maternal inherited in origin, that mean the duplication occur in the maternal allele of chromosome 15. Two families with interstitial duplication are identified in this study and the rest of the cases with genomic aberration that arise de novo. The clinical features of our 15q duplication cohort are summarised in Table 1. The facial profiles of patients in this study are shown in Figure 2.
The clinical features of our Chinese patients are compared with western populations in Table 2.7-17 It showed that most of clinical features in our Chinese cohort included dysmorphism, joint laxity, hypotonia, intellectual disability and autism are comparable with Caucasian. However, epilepsy is less common while squint is more prevalent in Chinese patients with 15q duplication syndrome.
15q duplication syndrome is a neurogenetic syndrome that frequently associated with neurodevelopmental disease. The prevalence of 15q11-q13 duplication in patients with autism is widely assumed to be 1-3% based on two early studies.5,6 With the advancement of genomic technology like aCGH and better understanding in the association of copy number variation (CNV) with human disease. It is now known that approximately 10-20% of patients with intellectual disability and 10% of autism or ASD patients have clinical significant CNVs.18,19 In the latest reviews and meta-analysis, it has found that 15q11-q13 duplication happened in 1% of patients with autism or ASD and 3% of patient with intellectual disability.20,21 The prevalence of 15q duplication in our Chinese cohort with intellectual disability and autism or ASD is 1.0% and 2.9%, respectively.
Concerning on the clinical phenotype, the most consistent features in our study are hypotonia and intellectual disability. About two third of them have facial dysmorphism and autism. All these are comparable with other studies in the literatures.7-17 Epilepsy occurs in 60% of 15q duplication syndrome in the latest survey by Dup 15 alliance with 80% has multiple seizure types and 40% has infantile spasm.22 In our cohort, only 33.3% (4/12) of them have epilepsy with tonic-clonic and focal seizure as the most common seizure semiology. Only one of our patients has infantile spasm with the onset at 3 months of age. Despite multiple anticonvulsant, his epilepsy is refractory that currently evolving into Lennox-Gastaut syndrome. For the rest of the patients, the epilepsy is well controlled by one anticonvulsant. Despite pseudosquint are common among Chinese, true squint happen in more than 90% of our patients that is statistically significant different from other ethnic groups.
Chromosome 15q11-q13 region imprinting patterns and its long range gene expression are mediated by imprinting control region (ICR) that located upstream of paternal transcription unit SNRPN gene through the UBE3A gene interaction.23 The imprinting and parent-of-origin effect has been well demonstrated by our 2 families with interstitial duplication. By methylation specific-MLPA and microstatellite study, the interstitial 15q 11.2 duplication in the mothers of family 6 and family 10 in our study have confirmed to locate at paternal and maternal allele of chromosome 15, respectively. The mother in family 6 with interstitial 15q11.2 duplication in the paternal allele has normal phenotype, while the mother in family 10 with inherited interstitial 15q11.2 duplication in the maternal allele has hypotonia and mild intellectual disability. Maternal duplication of the UBE3A gene is the proposed mechanism for such imprinting effect.24,25
For the genotype-phenotype correlation, a dosage effect on 15q11-q13 copies to clinical severity has been demonstrated in previous studies.13,16,26 The clinical presentation for isodicentric chromosome 15 with 4 copies of SNRPN gene is more severe than interstitial duplication with 3 copies of SNRPN gene. The mitigating effect of mosaic 15q duplication has also been reported which depend on the percentage and type of cell line involved. However, such correlation cannot be shown in our Chinese cohort due to small sample size. The phenotypic spectrum and severity among those isodicentric chromosome 15, interstitial duplication and mosaic isodicentric chromosome 15 are similar in this study.
Concerning on recurrence risk and genetic counselling, for isodicentric chromosome 15, it usually arise denovo that recurrence risk in family is negligible. For interstitial duplication, the inheritance risk to offspring from affected proband is 50%. Due to genomic imprinting, the phenotypic effect depends on the parent-of-origin. Duplication in the maternal allele of chromosome 15 has high risk of developing neurodevelopmental disease and epilepsy, while duplication in the paternal allele usually phenotypically normal.15,18
This 5 years' review is the first territory-wide study of 15q duplication syndrome in Chinese. It showed that the incidence and clinical features of Chinese 15q duplication are comparable with other western populations. The prevalence of 15q duplication in our Chinese cohort with intellectual disability and autism or autistic spectrum disease is 1.0% and 2.9%, respectively. Early diagnosis is important for managing 15q duplication patients as they have high risk of developing epilepsy and neurodevelopmental disorder that anticipatory guidance from different specialties is essential. Genetic confirmation on the underlying mechanism for 15q duplication is also important for reproductive risk assessment.
We thank all the paediatricians and physicians who have referred their 15q duplication syndrome patients to our service. We are also grateful to all the laboratory staff in CGS for their technical support.
Conflict of interest
We have no conflict of interest to declare.
1. Christian SL, Fantes JA, Mewborn SK, Huang B, Ledbetter DH. Large genomic duplicons map to sites of instability in the Prader-Willi/Angelman syndrome chromosome region (15q11-q13). Hum Mol Genet 1999;8:1025-37.
2. Robinson WP, Dutly F, Nicholls RD et al. The mechanisms involved in formation of deletions and duplications of 15q11-q13. J Med Genet 1998;35:130-6.
3. Battaglia A. The inv dup(15) or idic(15) syndrome: a clinically recognizable neurogenetic disorder. Brain Dev 2005;27:365-9.
4. Makoff AJ, Flomen RH. Detailed analysis of 15q11-q14 sequence corrects errors and gaps in the public access sequence to fully reveal large segmental duplications at breakpoints for Prader-Willi, Angelman and inv dup(15) syndromes. Genome Biol 2007; 8:R114.
5. Cook Jr EH, Courchesne RY, Cox NJ, et al. Linkage-disequilibrium mapping of autistic disorder, with 15q11-13 markers. Am J Hum Genet 1998;62:1077-83.
6. Schroer RJ, Phelan MC, Michaelis RC, et al. Autism and maternally derived aberrations of chromosome 15q. Am J Med Genet 1998;76:327-36.
7. Robinson WP, Binkert F, Gine R, et al. Clinical and molecular analysis of five inv dup (15) patients. Eur J Hum Genet 1993; 1:37-50.
8. Mignon C, Malzac P, Moncla A, et al. Clinical heterogeneity in 16 patients with inv dup 15 chromosome: cytogenetic and molecular studies, search for an imprinting effect. Eur J Hum Genet 1996;4:88-100.
9. Battaglia A, Gurrieri F, Bertini E, et al. The inv dup(15) syndrome: A clinically recognizable syndrome with altered behavior, mental retardation and epilepsy. Neurology 1997;48:1081-6.
10. Hou JW, Wang TR. Unusual features in children with inv dup(15) supernumerary marker: A study of genotype phenotype correlation in Taiwan. Eur J Pediatr 1998;157:122-7.
11. Webb T, Hardy CA, King M, Watkiss E, Mitchell C, Cole T. A clinical, cytogenetic and molecular study of ten probands with supernumerary inv dup(15) marker chromosomes. Clin Genet 1998;53:34-43.
12. Wolpert CM, Menold MM, Bass MP. Three probands with autistic disorder and isodicentric chromosome 15. Am J Med Genet 2000;96:365-72.
13. Battaglia A. The inv dup (15) or idic (15) syndrome (Tetrasomy 15q). Orphanet J Rare Dis 2008;3:30.
14. Borgatti R, Piccinelli P, Passoni D, et al. Relationship between clinical and genetic features in "inverted duplicated chromosome 15" patients. Pediatr Neurol 2001;24:111-6.
15. Crolla JA, Youings SA, Ennis S, Jacobs PA. Supernumerary marker chromosomes in man: Parental origin, mosaicism and maternal age revisited. Eur J Hum Genet 2005;13:154-60.
16. Dennis NR, Veltman MW, Thompson R, et al. Clinical findings in 33 patients with large supernumerary marker-(15) chromosomes and 3 patients with triplication of 15q11-15q13. AmJ Med Genet 2006;140:434-41.
17. Valente KD, Freitas A, Fridman C, et al. Inv dup (15): is the electroclinical phenotype helpful for this challenging clinical diagnosis? Clin Neurophysiol 2006;117:803-9.
18. Shishido E, Aleksic B, Ozaki N. Copy-number variation in the pathogenesis of autism spectrum disorder. Psychiatry Clin Neurosci 2014;68:85-95.
19. Miller DT, Adam MP, Aradhya S, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 2010;14;86:749-64.
20. Devlin B, Scherer SW. Genetic architecture in autism spectrum disorder. Curr Opin Genet Dev 2012;22:229-37.
21. Cooper GM, Coe BP, Girirajan S, et al. A copy number variation morbidity map of developmental delay. Nat Genet 2011;14;43:838-46.
22. Conant KD, Finucane B, Cleary N, et al. A survey of seizures and current treatments in 15q duplication syndrome. Epilepsia 2014; 55:396-402.
23. Hogart A, Wu D, LaSalle JM, Schanen NC. The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 2010;38:181-91.
24. Marshall CR, Noor A, Vincent JB, et al. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet 2008; 82:477-88.
25. Szatmari P, Paterson AD, Zwaigenbaum L, et al. Mapping autism risk loci using genetic linkage and chromosomal rearrangements. Nat Genet 2007;39:319-28.
26. Schinzel AA, Brecevic L, Bernasconi F, et al. Intrachromosomal triplication of 15q11-q13. J Med Genet 1994;31:798-803.