Reciprocal Microduplication of the Williams-Beuren Syndrome Chromosome Region in a 9-Year-Old Omani Boy

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1 Reciprocal Microduplication of the Williams-Beuren Syndrome Chromosome Region in a 9-Year-Old Omani Boy Shruthi Mohan, MSc, 1 Sheela Nampoothiri, DNB, MSc, 2 Dhanya Yesodharan, MD, 2 Vettriselvi Venkatesan, PhD, 1 Teena Koshy, MSc, 1 Solomon F.D. Paul, PhD, 1 Venkatachalam Perumal, PhD 1 * Laboratory Medicine 47:2: ABSTRACT Background: Microdeletions of the 7q11.23 Williams-Beuren syndrome chromosome region (WBSCR) are reported with a frequency of 1 in 10,000, whereas microduplications of the region, although expected to occur at the same frequency, are not widely reported. Method: We evaluated a 9-year old Omani boy for idiopathic intellectual disability using genetic methods, including multiplex ligation-dependent probe amplification (MLPA), for detection of microdeletions (P064-B3). Results: MLPA analysis revealed that the boy has a rare microduplication of the WBSCR. Prominent clinical features include global developmental delay with pronounced speech delay, dysmorphic facies, and autistic features. Conclusion: Microduplications, in general, are reported at a lesser frequency, perhaps owing to their milder phenotype. Complete genetic assessment in children with idiopathic intellectual disability would help in identifying rare conditions such as duplication of the WBSCR. Keywords: microduplication, 7q11.23, Williams-Beuren syndrome, speech delay, intellectual disability, multiplex ligation-dependent probe amplification Subtle chromosomal rearrangements are associated with a number of intellectual disability (ID) related syndromes. Recently, various interstitial and telomeric rearrangements of chromosomes have become well-characterized etiological factors of ID. 1,2 These microdeletions and microduplications, also known as copy-number variations, are mediated by lowcopy repeats present in approximately 5% of the human genome; 3,4 these entities play a significant role in disease mechanisms by altering the dosage of functional genes. Abbreviations ID, intellectual disability; WBS, Williams-Beuren syndrome; WBSCR, Williams-Beuren syndrome chromosome region; NAHR, nonallelic homologous recombination; MLPA, multiplex ligation-dependent probe amplification; MRI, magnetic resonance imaging; EEG, electroencephalogram; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction; ADHD, attention-deficit hyperactivity disorder; CAP-Gly; ELN, elastin; LMK1, LIM kinase 1; STX1A, syntaxin 1 Department of Human Genetics, Sri Ramachandra University, Chennai, India and 2 Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Center, Kochi, India *To whom correspondence should be addressed. venkip@yahoo.com Williams-Beuren syndrome (WBS) is one such microdeletion syndrome, with an incidence of approximately 1 in 10,000 live births. 5 Although WBS is often associated with a microdeletion in the WBS chromosome region (WBSCR), there been a few reports of microduplications in the region. 6-8 In most individuals with a rearrangement in the WBSCR, the region involved in rearrangement is 1.5 Mb in length, encompassing about 26 to 28 genes, between the NSUN5 gene on the centromeric end and GTF2IRD2 gene on the telomeric end. 9 Breakpoints vary depending on the extent of rearrangement; however, commonly reported breakpoints lie within the GTF2IP1 and GTF2I genes. 10 The phenotypic features of the deletion and duplication syndromes vary, although they occur due to the same molecular mechanism of nonallelic homologous recombination (NAHR). 11,12 NAHR results from misalignment due to similarity among low-copy repeats, which are abundant in the pericentromeric and subtelomeric regions. 13 Herein, we describe the case of a 9-year-old Omani boy with ID and dysmorphic features, suggestive of chromosomal imbalance as a possible etiology. Multiplex ligation-dependent probe amplification (MLPA) analysis revealed a rare duplication VC American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com 171

2 in the WBSCR. We discuss the observed features of this case in connection with available background information on the syndrome. Case Report The proband was a 9-year-old Omani boy born to a fourthdegree consanguineous couple; he had weighed 3 kg at birth. He was the firstborn; his 2 younger siblings did not have any of the health problems that he had experienced. The boy had had 4 episodes of seizures, with uprolling of eyes that lasted for approximately 15 minutes, at the age of 5 months. Occasional blackouts while playing had also been observed by his parents. He showed overall developmental delay, with prominent speech delay disproportionate to his motor delay. The boy had started to speak only at age 5 years, and at age 9 years, he was still not toilet trained. His height was 132 cm (25th to 50th percentile), his weight was 31 kg (50th to 75th percentile), and his head circumference was 51.5 cm (10th to 25th percentile). The age of his bones was delayed and corresponded to 7 years, despite that his chronological age was 9 years. He walked with a broadbased gait. He had a cheerful and pleasant personality but was disinterested in playing with other children. Results of ultrasound evaluation of the abdomen, and magnetic resonance imaging (MRI) of the brain and electroencephalogram (EEG) of the proband were normal. A detailed physical examination revealed dysmorphic features, including macrostomia (Image 1A), prominent nose with overhanging nasal tip low-set flared pinna (Image 1B), and down slanted eyes. Other prominent features included fair complexion, brachydactyly of fingers (Image 1C), and bilateral partial cutaneous syndactyly between the second and third toes. He had a right external angular dermoid, which was excised. He showed a few autistic features, and therefore, his specimen was tested for Fragile X syndrome and Angelman syndrome. Further evaluation by karyotyping, fluorescence in situ hybridization (FISH), and MLPA was performed. subtelomeric rearrangements. For the MLPA analysis, DNA was isolated using the QIAGEN QIAamp blood mini kit (QIAGEN Inc) from approximately 2 ml of peripheral blood obtained from the proband. The SALSA MLPA probe kit P064 Version B3 (MRC- Holland) was used to evaluate the role of chromosomal rearrangements as the cause of intellectual disability in the proband. The P064 kit contained probes for the following genomic regions: 1p36 telomere, 5q35.3 NSD1 gene for Sotos syndrome, 7p21.2 TWIST1 and TWISTNB for Saethre-Chotzen syndrome, 7q11.23 for the WBS region, 15q11.2 for the Prader- Willi syndrome region, 17p11.2 region for Smith-Magenis syndrome, 17p13.3 for the Miller-Dieker syndrome region, 20p12.2 JAG1 gene for Alagille syndrome, and 22q11.21 for the DiGeorge syndrome region. Three normal DNA specimens from age-matched male donors were included as control specimens. DNA was denatured and the probes hybridized overnight and ligated, after which the bound probes were amplified. Fragment separation of polymerase chain reaction (PCR) products was performed in the ABI 3730 DNA analyzer (Life Technologies) and the data analyzed using the GeneMarker software, Version (SoftGenetics, LLC). Results The results of karyotype analysis showed a normal karyotype of 46,XY. The results of the southern blot study for Fragile X and methylation study for Angelman syndrome were normal. FISH testing for subtelomeric rearrangements also yielded a negative result, suggesting the absence of telomeric structural rearrangements. The MLPA peaks were analyzed and the fluorescence intensities were compared between test and control specimens. The results were interpreted based on fluorescence intensity ratios. All 6 probes for the genes, CLIP2 (2 probes),fzd9, ELN, LIMK1, andstx1a in the 1 Mb region within the WBSCR showed ratios greater than 1.3, indicating duplication of the WBS region (Image 2). Materials and Methods Genetic testing was performed on the boy after obtaining informed consent from his parents. FISH was performed using the Vysis ToTelVysion kit (Abbott Laboratories, Inc) on chromosomes obtained from cultured lymphocytes, to rule out Discussion Less than 100 cases of microduplication of the WBSCR have been described in the literature. 14 Although the prevalence of the WBSCR duplication is estimated to be similar to that of the WBS microdeletion, the population frequency is not 172 Lab Medicine 2016;47:2;

3 Image 1 Facial/physical features of proband, a 9-year-old Omani boy. A, Macrostomia. B, Low-set flared pinna and prominent nose with overhanging nasal tip. C, Brachydactyly of fingers. clearly established, due to paucity of data. However, the cases of a few individuals with the duplication but no apparent phenotypic abnormality have been described. 8,15 Although the clinical phenotype of individuals with WBSCR duplication is not completely delineated, it is agreed that the features are milder and less distinct in comparison to the deletion syndrome. 16 Reported clinical features of individuals with a duplication of the WBSCR region include broad forehead; high, broad nose; short philtrum; thin lips; normal growth; congenital heart defects; joint laxity; hypotonia; seizures; nonspecific brain abnormalities observable on MRI; developmental delay; intellectual delay; speech and language delay; deficits of social interaction/aggressive behavior; autism spectrum behavior; and attention deficit hyperactivity disorder (ADHD) with sparing of visuospatial skills. 16 It is widely reported colloquially that speech delay is the most distinctive clinical finding in patients with WBSregion microduplication. Severe speech delay has been observed in the case of triplication of the region, adding evidence to the association. Similarly, in this case report, the Lab Medicine 2016;47:2;

4 Image 2 Analysis image of multiplex ligation-dependent probe amplification (MLPA) result, showing duplication of probes (arrows): CLIP2, FZD9, ELN, LIMK1, and STX1A in the 7q11.23 Williams-Beuren syndrome chromosome region (WBSCR). proband showed a significant delay in speech. Contrary to the findings of deficits in social behavior in children with the duplication, our proband had a cheerful personality. Some reports suggest that social interaction had improved with familiarization in the affected children. 14 The brain MRI findings were normal for the proband. Compared with WBSCR deletion, individuals with the WBS microduplication show a milder phenotype. 16 Dysmorphic features of the mouth vary between the deletion and duplication syndromes: individuals with the deletion have a long philtrum and full lips, whereas those with duplication have a short philtrum with thin lips. 17 Growth may be retarded in the case of deletions, whereas individuals with duplication may show normal growth. Seizures are more common in individuals with duplications, along with hypotonia, which is also observed in patients with deletion. Cardiac abnormalities are less common in duplications, whereas supravalvular aortic stenosis is frequently observed in individuals with WBSCR deletion. Behavioral differences are stark between individuals with deletions and those with duplications. Although individuals with the deletion are excessively social, those with duplications show restrained social interaction, complicated by their profound speech and language delays. Many genes, such as frizzled drosophila homolog of 9 (FZD9), STX1A, ELN, CAP-Gly domain containing linker protein 2 (CLIP2), LIMK1, EIF4H, LAT2, RFC2, and GTF2IRD1 have been implicated in the pathogenesis of WBS-region deletion and duplication syndromes. Our proband was found to have duplications of FZD9, STX1A, ELN, CLIP2, and LIMK1 genes (those covered in the probe kit). The implication of the increased dosage of these genes is not clearly understood; however, those genes have been extensively studied in the case of deletions. CLIP2 is involved in the regulation of microtubule dynamics 18 and is suggested to contribute to the motor and cognitive aspects of WBS deletion. 19 FZD9 is reported to play a role in neurodevelopment and behavior; 20 however, its involvement in disease pathogenesis is poorly understood. The neurodevelopmental manifestations observed with the duplication may be attributed to the excess of these gene products. Elastin (ELN) is a major component of the elastic 174 Lab Medicine 2016;47:2;

5 fibers that strengthen connective tissue throughout the body. Its haplo insufficiency is reported colloquially to cause supravalvular aortic stenosis in patients with WBS. Because heart defects are less common in patients with duplications, the significance of duplication of this gene is not well understood. LIM kinase 1 (LIMK1) is a serine protein kinase involved in the organization of the actin cytoskeleton 21 and is thought to have a functional role in visuospatial constructive cognition. 22 Syntaxin (STX1A) plays a key role in ion-channel regulation and synaptic exocytosis; 23 its dosage alteration may lead to impaired glucose tolerance. 24 The implication of these genes in the phenotype of individuals with the duplication is unclear because visuospatial skills and glucose metabolism are not affected in the duplication. 16 The opposing phenotypes of the deletion and duplication syndromes suggest that genes in the region are highly dosage sensitive. 25 However, more investigation on the effects of increased dosage of these genes is essential to better correlation of the genotype and phenotype. This report contributes to the existing information in the literature on the phenotype of WBSCR duplications, although the lack of availability of more clinical and technical data poses a limitation to the study. More research on affected individuals with the WBSCR duplications can aid in better inference of the genotype-phenotype correlation. Because the occurrence and diagnosis of WBS duplication are infrequent, insights from every case are essential to expand the knowledge on this disorder and to enable more rapid, more efficient diagnosis in the future. LM References 1. Biesecker LG. The end of the beginning of chromosome ends. Am J Med Genet. 2002;107: Tyson C, Harvard C, Locker R, et al. Submicroscopic deletions and duplications in individuals with intellectual disability detected by array-cgh. Am J Med Genet. 2005;139A: Bailey JA, Gu Z, Clark RA, et al. Recent segmental duplications in the human genome. Science. 2002;297: Mc Carroll SA, Kuruvilla FG, Korn JM, et al. 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