Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits

European Journal of Human Genetics (2014) 22, 64–70 & 2014 Macmillan Publishers Limited All rights reserved 1018-4813/14 www.nature.com/ejhg ARTICLE Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits Carmela Fusco1,6, Lucia Micale1,6, Bartolomeo Augello1, Maria Teresa Pellico1, Deny Menghini2, Paolo Alfieri2, Maria Cristina Digilio3, Barbara Mandriani1,4, Massimo Carella1, Orazio Palumbo1, Stefano Vicari2 and Giuseppe Merla*,1,5 Williams Beuren syndrome (WBS) is a multisystemic disorder caused by a hemizygous deletion of 1.5 Mb on chromosome 7q11.23 spanning 28 genes. A few patients with larger and smaller WBS deletion have been reported. They show clinical features that vary between isolated SVAS to the full spectrum of WBS phenotype, associated with epilepsy or autism spectrum behavior. Here we describe four patients with atypical WBS 7q11.23 deletions. Two carry B3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG) genes. Other two carry a shorter deletion of B1.2 Mb at centromeric side that excludes the distal WBS genes BAZ1B and FZD9. Along with previously reported cases, genotype–phenotype correlation in the patients described here further suggests that haploinsufficiency of HIP1 and YWHAG might cause the severe neurological and neuropsychological deficits including epilepsy and autistic traits, and that the preservation of BAZ1B and FZD9 genes may be related to mild facial features and moderate neuropsychological deficits. This report highlights the importance to characterize additional patients with 7q11.23 atypical deletions comparing neuropsychological and clinical features between these individuals to shed light on the pathogenic role of genes within and flanking the WBS region. European Journal of Human Genetics (2014) 22, 64–70; doi:10.1038/ejhg.2013.101; published online 12 June 2013 Keywords: Williams Beuren syndrome; 7q11.23; haploinsufficiency; qPCR INTRODUCTION The 1.5–1.8 Mb hemizygous deletion of roughly 28 annotated genes at 7q11.23 region causes Williams Beuren syndrome (WBS; OMIM number 194050). WBS patients display a characteristic pattern of symptoms including typical facial dysmorphism, supravalvular aortic stenosis (SVAS), weakness of connective tissue, short stature, mild-tomoderate intellectual disability (ID), and a characteristic cognitive profile that includes relative strengths in verbal short term memory and lexical comprehension, alongside severe weakness in visuospatial construction. Other clinical features include growth retardation, hyperacusis, premature ageing, hypercalcemia, glucose intolerance, renal anomalies, dental defects, gastrointestinal problems, urinary tract abnormalities, weakness in daily living skills, and motor abilities.1 The dissection of the WBS phenotype relies mainly on evidences from functional studies of single genes, animal models, and analysis of WBS individuals with atypical deletions. These studies suggest correlations between haploinsufficiency of some WBS genes and WBS phenotypic features. For instance, reduced BAZ1B protein level has been linked to cardiac, craniofacial, and hypercalcemia defects; hemizygosity of LIMK1, CLIP2, GTF2IRD1, and GTF2I is associated to the specific WBS cognitive profile and craniofacial features.2,3 Here we describe four patients with atypical 7q11.23 deletions and complex WBS phenotypes. Two carry B3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG). The other two carry shorter deletions of B1.2 Mb at centromeric side that do not include the more distal WBS genes. SUBJECTS AND METHODS Subjects and clinical evaluation Blood samples were obtained from proband and available parents after acquiring informed consent approved by the institutional review board. In WBS154 and WBS166 patients, the global cognitive evaluation was conducted by the Wechsler Intelligence Scale for Children Third Edition (WISC-III).4 As WBS160 had a severe ID and WBS179 was very young, both were not able to complete the WISC-III, and therefore their global cognitive abilities were assessed by using the Leiter International Performance Scale-Revised5 and the Griffiths Scales of Mental Development),6 respectively. 1Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy; 2Child NeuroPsychiatry Unit, Neuroscience Department, IRCCS Children Hospital Bambino Gesù, Rome, Italy; 3Medical Genetics, IRCCS Children Hospital Bambino Gesù, Rome, Italy; 4PhD Program, Biomedical Sciences and Translational Medicine, University of Brescia, Brescia, Italy; 5PhD Program, Reproduction and Development Sciences, University of Trieste, Trieste, Italy 6These authors are joint first authors. *Correspondence: Dr G Merla, Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, Poliambulatorio Giovanni Paolo II, San Giovanni Rotondo (FG) I-71013, Italy. Tel: +39 0882 416350, Fax: +39 0882 411616, E-mail: Received 12 December 2012; revised 6 March 2013; accepted 10 April 2013; published online 12 June 2013 Atypical patients in Williams Beuren syndrome C Fusco et al 65 Complete evaluation concerning linguistic (production and comprehension), visuo-motor, praxic, coordination, adaptive abilities, and behavior aspects was reported for all the patients (Table 1). Multiple-ligation probe amplification and quantitative real-time PCR The multiple-ligation probe amplification (MLPA) analysis was performed with SALSA kit P029 (MRC Holland, http://www.mlpa.com), according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was carried out as described,3 with oligo pairs reported in Supplementary Table S2 and based on the UCSC GRCh37/hg19 assembly. CytoScan HD array and junctions fragment mapping The CytoScan HD assay was performed according to the manufacturer’s protocol. Briefly, genomic DNA was digested, ligated to an adapter, and subjected to PCR amplification. After DNase I digestion, PCR products were labeled and hybridized. After that washing and staining were performed using the Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA). The array was then scanned with the Scanner 3000 7G (Affymetrix), and both quality control step and copy number analysis were performed using the Chromosome Analysis Suite Software (Affymetrix): (i) the raw data file (CEL) was normalized using the default options; (ii) an unpaired analysis was performed using as baseline the 270 HapMap samples in order to obtain copy numbers value from CEL files while the amplified and/or deleted regions were detected using a standard Hidden Markov Model method. The Database of Genomic Variants (http:// www.projects.tcga.ca/variation/ (...truncated)